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Apoptosis

From Genes to Proteins to Cells

BD Biosciences
Clontech
Discovery Labware
Immunocytometry Systems
Pharmingen

Table of Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Gene Expression Analysis . . . . . . . . . . . . . . 2
2.1. RiboQuant Multi-Probe Ribonuclease
Protection Assay System . . . . . . . . . . . . . . 2-6
2.1.1. Custom RiboQuant RNase
Protection Assays . . . . . . . . . . . . . . . . 7
2.2. Atlas Arrays . . . . . . . . . . . . . . . . . . . . . 8-10

4. Apoptosis Detection and Functional


Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.1. Antibodies to Active Caspase-3 . . . . . . . . . 35
4.1.1. Monitoring Caspase-3 Activation during
Apoptosis using a novel
ELISA System . . . . . . . . . . . . . . . . . . . 36-37
4.1.2. Human Active Caspase-3 Cytometric
Bead Array (CBA) Kit . . . . . . . . . . . . . 38

3. Protein Expression Analysis . . . . . . . . . . . 13

4.2. Caspase Assays, Active Human Caspases,


Substrates, Inhibitors,
Inducers and Sets . . . . . . . . . . . . . . . . . 39-42

3.1. Death Receptors and Ligands . . . . . . . . 14-16

4.2.1. Caspase3-Sensor Vector . . . . . . . . . . 43

3.2. Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.3. ApoAlert Bid Vectors:


DsRed2 & EGFP . . . . . . . . . . . . . . . . . . . 44-45

2.3. HeLa & MCF7 Apoptosis cDNA Panels . . 11-12

3.2.1. Antibodies to Various Caspases . . 18-19


4.4. ApoAlert Glutathione Detection Kit. . . . 46
3.2.3. Antibodies to Caspase-3 Protein
Substrates. . . . . . . . . . . . . . . . . . . 20-21

4.5. ApoAlert Cell Fractionation Kit . . . . . . . 47

3.3. Bcl-2 Family Members . . . . . . . . . . . . . . 22-23

4.6. ApoAlert Mitochondrial Membrane


Sensor Kit . . . . . . . . . . . . . . . . . . . . . . . . . . 48

3.4. Cytochrome c. . . . . . . . . . . . . . . . . . . . . 24-25


4.7. Annexin V Reagents and Kits . . . . . . . . 49-51
3.5. Other Apoptosis-Related Proteins . . . . . . . 26
3.5.1. Signal Transducers . . . . . . . . . . . . 26-27

4.8. ApoAlert Nitric Oxide/Annexin V Dual


Sensor Kit . . . . . . . . . . . . . . . . . . . . . . . 52-54

3.5.2. Tumor Suppressors . . . . . . . . . . . . 28-29

4.8. BD Biosciences TUNEL Assays . . . . . . . . . . . 55

3.6. Positiv Control Lysates . . . . . . . . . . . . . . . . 30

4.8.1. APO-DIRECT and


APO-BRDU Kits . . . . . . . . . . . . . . . . 55

3.7. Recombinant Proteins and Peptides. . . . . . 30


3.8. Protein Expression Screening . . . . . . . . . . . 31
3.8.1. Antibody Sampler Kits. . . . . . . . . . . . 31
3.8.2. BD PowerBlot . . . . . . . . . . . . . . . . 32-33

4.8.2. ApoAlert DNA Fragmentation


Assay Kit . . . . . . . . . . . . . . . . . . . 56-57
4.10. ApoAlert LM-PCR Ladder
Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . 58-59

3.8.3. Customize Your BD PowerBlot . . . 34

BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. 2002 BD

www.bdbiosciences.com

Abbreviations
AFC
AMC
APC
Apps.
DsRed
E
EBS
EGFP
EYFP
FA
FBS
FC
FITC
fmk
GSH
Hu
IF
IHC(F)
IHC(P)
IP
IVK
LM-PCR
Ms
MTC
NA/LE
NES
NLS
NO
PI
PE
pNA
PS
R
RPA
RXNS
SF
TUNEL
WB

7-amino-4-trifluoromethy
coumarin
7-amino-4-methoxy coumarin
Allophycocyanin
Applications
Discosoma sp. Red Fluorescent
Protein
ELISA
Epitope Blocking Studies
Aequorea victoria Enhanced
Green Fluorescent Protein
Aequorea victoria Enhanced
Yellow Fluorescent Protein
Functional Assays
Fetal Bovine Serum
Flow Cytometry
Fluorescein isothiocyanate
Fluoromethylketone
Glutathione
Human
Immunofluorescence
Immunohistochemistry (Frozen)
Immunohistochemistry (Paraffin)
Immunoprecipitation
In Vitro Kinase Assay
Ligation-mediated polymerase
chain reaction
Mouse
Multiple Tissue cDNA
No Azide/Low Endotoxin
nuclear export signal
nuclear localization signal
Nitric oxide
Propidiumiodide
Phycoerythrin
p-Nitroanilide
Phosphatidylserine
Rat
Ribonuclease Protection Assay
Reactions
Spectrofluorometry
terminal deoxynucleotidyl
transferase-mediated dUTP
nick-end-labeling
Western Blot

Introduction
Programmed cell death is a normal
physiological process which occurs
during embryonic development as
well as in maintenance of tissue
homeostasis. Kerr et al. originally
described two forms of cell death
which may occur in the absence of
pathological manifestations, necrosis and apoptosis1. The term apoptosis, from the Greek word for
falling off of leaves from a tree, is
used to describe a process in which
a cell actively participates in its own
destructive processes. The apoptotic
program is characterized by certain
morphological features. These
include changes in the plasma membrane such as loss of membrane
asymmetry and attachment, a condensation of the cytoplasm and
nucleus, and internucleosomal
cleavage of DNA. In the final
stages, the dying cells become fragmented into apoptotic bodies
which are rapidly eliminated by
phagocytic cells without eliciting
significant inflammatory damage to
surrounding cells. Necrosis, which
typically occurs as a result of cell
injury or exposure to cytotoxic
chemicals, is distinct from apoptosis
in both morphological and biochemical characteristics. Necrotic
cell death begins with swelling of
the cell and mitochondrial contents,

followed by rupture of the cell


membrane2. In contrast to apoptosis, necrosis can trigger an inflammatory reaction in the surrounding
tissue as a result of cytoplasmic
contents, many of which are proteolytic enzymes.
Inappropriate induction of apoptosis has broad ranging pathologic
implications and has been associated with Alzheimerss3, Hodgkins4
and graft-versus-host diseases,
transplant rejection5, and autoimmune disorders such as cancer6 and
AIDS7. As a result, the apoptosis
research field is explosive, with
publications related to apoptotic
processes numbering in the thousands every year. BD Biosciences
with its business units Clontech,
Discovery Labware,
Immunocytometry Systems, and
Pharmingen offers a broad range of
Apoptosis related products and systems. We offer total solutions from
consumable reagents and cell culture ware to instrumentation, from
standard catalog products to customized services. With our Genes to
Proteins to Cells approach, we provide tools for gene and protein
expression analysis as well as for
functional assays.

References
1. Kerr, J.F.R., A.H. Wyllie and A.R. Curie. 1972. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26:239-257
2. Majno, G. and J. Joris. 1995: Apoptosis, oncosis and necrosis. An overview of cell death. Am. J. Pathol.
146:3-16.
3. Kitumura, Y., Perry, P.J. Whitehouse and T. Taniguchi. 1998. Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x,
Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimers disease. Brain Res. 780:260-269.
4. Lorenzen, J., J. Thiel and R. Fischer. 1997. The mummified Hodgkin cell: Cell death in Hodgkins disease. Am. J. Pathol.
182:288-298
5. Lin, T., Brunner, B. B. Tietz, J. Madsen, E. Bonfoco, M. Reaves, M. Hufet and D.R.Green. 1998. Fas ligand-mediated
killing by intestinal intraepithelial lymphocytes. Participation in intestinal graft-versus-host disease. J. Clin. Invest. 101:5705747
6. Thomson, C.B. 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267:1456-1462.
7. Wang, L., G.R. Klimpel, J.M. Planas, H. Li and M.W. Cloyd. 1998. Apoptotic killing of CD4+ T lymphocytes in HIV-1
infected PHA-stimulated PBL cultures is mediated by CD8+ LAK cells. J. Virol. 241:169-180

www.bdbiosciences.com

hAPO-2
1
bcl-x

L,S

bcl-x

bfl1
bfl1
bik
bak

2. Gene Expression Analysis

bik

bax
bak
bcl-2

bax

mcl-1
bcl-2

2.1. RiboQuant Multi-Probe Ribonuclease Protection


Assay System

mcl 1

L32

PBMC

PBMC

Stimulated

Jurkat

Stimulated

Probe

Jurkat

GAPDH

hAPO-3
1

Caspase-8
FASL
FAS

FASL

FADD

FAS

DR3
FAP

DR3

FAF
TRAIL

TRAIL

TNFRp55
TRADD
RIP

TNFRp55
TRADD

L32

Stimulated

PBMC

PBMC

Jurkat

Stimulated

Probe

Jurkat

GAPDH

Autoradiograms of protection assays


utilizing hAPO-2 and hAPO-3 multiprobe template sets with RNA isolated
from Jurkat T cells and Human PBMCs.
Jurkat cells and human PBMCs were cultured in the presence or absence of
1 g/ml of ionomycin for 3.5 hr. Total
cellular RNA was isolated and treated
according to protocol using hAPO-2
(Cat. No. 556191; bcl-2 related molecules) and hAPO-3 (Cat. No. 556192;
death receptor signaling molecules).
The profile from PBMCs of expressed
RNAs was consistent with activated and
proliferating
cells.
Characteristic
increases in the level of anti-apoptotic
messages were seen for bcl-2, bcl-x, bfl1, mcl-1, and FAP. The levels of transcripts coding for molecules responsible
for causing death were generally
reduced (see bak, bax, DR3, TNFRp55,
and TRADD). One exception to this is
the increase in FasL expression. An
upregulation in FasL levels upon activation has been previously reported. The
combination of reduction in deathinducing molecules and the increase in
those which prevent death shifts the
balance in the cell to a state in which it
can grow and proliferate rather than
die.

The RiboQuant Multi-Probe


RNase Protection Assay (RPA) can
be used to measure mRNA levels of
molecules participating in apoptosis
during development, homeostasis,
and in many disease conditions. The
RPA is a method for detecting and
quantifying the expression of multiple genes in a single RNA sample.
This assay system involves the
hybridization of the RNA of interest
to their complementary antisense
RNA probes. Each individual apoptosis, cell cycle, tumor suppressor,
and stress protein gene specific template has been assembled into relevant sets, to be used by investigators
for the T7 RNA polymerase-directed
synthesis of a high-specific activity,
-32P-labeled, anti-sense RNA probe
set. In each set, we have included the
L32 gene (which encodes a ribosomal protein) and the GAPDH gene
to serve as housekeeping gene controls. Following transcription using
the
RiboQuant
In
Vitro
Transcription Kit (Cat. No. 556850)
the probe set and total target RNA
are solution hybridized overnight
using the reagents included in the
RPA Kit (Cat. No. 556134).
Subsequently, free probe and singlestranded non-protected RNA molecules
are
digested
with
a
pre-optimized mixture of RNase A
and T1. The remaining RNase-protected probes are treated with
Proteinase K, extracted, precipitated,
washed and loaded onto a 5% denaturing polyacrylamide gel. The
resulting resolved bands are separated on the gel according to their
size, and imaged by autoradiography, beta-scanning or phosphorimaging systems. The quantity of
each mRNA species in the original

www.bdbiosciences.com

RNA sample can then be determined


based on the intensity of the appropriately-sized, protected probe fragment.

Utility of the RPA for


Analyzing RNA from Cell
Populations
The BD RiboQuant Multi-Probe
RPA system is a powerful tool used
to detect and quantitate up to 13
mRNA species simultaneously in a
single sample of total RNA. BD
Biosciences Pharmingens strategy
for the development of multi-probe
RPA systems is to generate a series of
templates, each of distinct length and
each representing a sequence in a distinct mRNA species. The templates
are assembled into biologically relevant sets to be used by investigators
for the T7 polymerase-directed synthesis of a high-specific-activity,
radiolabeled anti-sense RNA probe
set.

Traditonally, probes have been generated using 32P-UTP, providing the


greatest sensitivity and signal intensity. However, the use and disposal
of radiolabeled nucleotides raises
issues: continued exposure of personnel to radioactivity, environmental concerns, and regulatory
requirements.
Now, researchers can take advantage
of the power and versatility of the
BD RiboQuant RPA System using
non-radiolabeled probes. Probes are
generated from the same multi-probe
template system, using biotin-labeled
UTP, instead of 32P-UTP. The protected probes are resolved on the
same denaturing polyacrylamide gel,
then transferred to a positivelycharged nylon membrane. Protected
probe signal is generated using
enhanced chemiluminescence, and

Day 1:

Probe Synthesis

detected by brief exposure to film.


The BD RiboQuant Non-Rad In
Vitro Transcription Kit is optimized
for the efficient synthesis of high-specific-activity, biotin-labeled riboprobes from the BD Biosciences
Pharmingen Multi-Probe Template
Sets. Each kit contains sufficient
reagents for 5 transcription reactions, yielding ~10 g biotin-labeled
probe per reaction.
The BD RiboQuant Non-Rad
Detection Kit includes 10 positivelycharged nylon membranes and all
reagents necessary for signal development using enhanced chemiluminescence. All protocols are described
in the newest edition of the BD
RiboQuant Instruction Manual.

RNA Preparation

Overnight Hybridization

Day 2:

RNase Treatment and


Purification of Protected Probes

Gel Preparation

Electrophoresis on Denaturing
Polyacrylamide Gel

Transfer to membrane
Chemiluminescent signal detection
Expose membrane to film

References for Apoptosis related template sets:


hAPO-3
Craxton A., Shu G., Graves J.D., Saklatvala J., Krebs E.G., Clark E.A. 1998. p38 MAPK is required for CD40-induced gene
expression and proliferation in B lymphocytes. J Immunol 161(7):3225-36
hAPO-5
Wajant H, Johannes FJ, Haas E, SiemienskiK, Schwenzer R, Schubert G, Weiss T, Grell M. Scheurich P., 1998.
Dominant-negative FADD inhibits TNFR60-, Fas/Apol 1- and TRAIL-R/Apo2-mediated cell death but not gene
induction. Curr Biol 8(2):113
Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS. 1998. NF-kappaB antiapoptosis: induction of
TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 81(5383):1680-3

www.bdbiosciences.com

Product Listing
Description

Size

BD RiboQuant In Vitro Transcription Kit


25 transcriptions
Ammonium Acetate
1.3 ml
DTT
50 l
EDTA
650 l
GTP/ATP/CTP/UTP Pool
25 l
RNase-Free DNase
50 l
RNAsin
25 l
T7 RNA Polymerase
25 l
Transcription Buffer
100 l
Yeast tRNA
50 l
BD RiboQuant Non-Rad In Vitro Transcription Kit NEW
5x Nucleotide Mix
5x Transcription Buffer
DTT
Enzyne Mix (RNasin & T7 RNA Polymerase)
RNase-free DNase I
EDTA
Glycogen
LiCl
RPA Kit
200 RPA reactions
Ammonium Acetate
24 ml
Hybridization Buffer
3.6 ml
Loading Buffer
1.3 ml
Proteinase K
300 l
Proteinase K Buffer
3.9 ml
RNase A + T1 Mix
60 l
RNase Buffer
25 ml
Yeast tRNA
300 l
BD RiboQuant Non-Rad Detection Kit NEW
Nylon Membrane
Hybridization Buffer
Hybridization Stringency Wash Buffer
Membrane Blocking Buffer
Wash Buffer (4x)
Substrate Equilibration Buffer
Streptavidin-Horseradish Peroxidase
Stable Peroxide Buffer
Luminol/Enhancer
BD RiboQuant RPA Starter Package
1 Kit
In Vitro Transcription Kit
25 transcriptions
RPA Kit, One free choice of RiboQuant Template Set
200 RPA reactions
BD RiboQuant Non-Rad RPA Starter Package
1 Kit
BD RiboQuant Non-Rad, In Vitro Transcription Kit
RPA Kit
200 RPA reactions
BD RiboQuant Non-Rad Detection Kit, One free choice of RiboQuant Template Set

Cat. No.

New Cat. No.

45004K
45013Z
45006Z
45012Z
45005Z
45010Z
45008Z
45009Z
45007A
45011Z

556850
556133
556126
556132
556125
556130
556128
556129
556127
556131
551917

45014K
45021Z
45015A
45022A
45019Z
45018A
45017Z
45016A
45020Z

556134
556141
556135
556142
556139
556138
556137
556136
556140
551918

45024K
45004K
45014K

556144
556850
556134

45014K

551917
556134
551918

Human Apoptosis-Related Template Sets

hAPO-1b
hAPO-1c
hAPO-2b
hAPO-2c
hAPO-3
hAPO-3b
hAPO-3d
hAPO-4
hAPO-5
hAPO-5b
hAPO-5c
hAPO-6

10
10
10
10
10
10
10
10
10
10
10
10

transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription

reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions

45378P
45607P
45614P
45609P
45131P
45611P
45616P
45141P
45151P
45610P
45613P
45612P

556209
556233
556240
556235
556163
556237
557278
556164
556165
556236
556239
556238

10
10
10
10

transcription
transcription
transcription
transcription

reactions
reactions
reactions
reactions

45358P
45354P
45355P
45601P

556195
556191
556192
556227

Mouse and Rat Apoptosis-Related Template Sets

mAPO-1
mAPO-2
mAPO-3
rAPO-1

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BD Pharmingen

Product Listing
Template

Protected

hAPO-1b

Protected

hAPO-1c

Caspase-8
Granzyme B
Caspase-3
Caspase-6
Caspase-5
Caspase-2 (S)
Caspase-7
Caspase-1
Caspase-2 (L)
Caspase-9
-L32 112
GAPDH

406
361
321
283
227
202 (S)
181
163
150 (L)
127
--

Template

Template

Protected

hAPO-2b

96

Caspase-8
Caspase-4
Caspase-3
Caspase-6
Caspase-10a
Caspase-5
Caspase-2 (S)
Caspase-7
Caspase-1
Caspase-2 (L)
Caspase-9
L32
GAPDH

406
361
320
283
253
227
203 (S)
181
163
150
127
113
96

bcl-W
bcl-X (L)
bcl-X (S)
bfl-1
BID
bik
bak
bax
bcl-2
mcl-1
-L32
GAPDH

401
364
324
286
257
226
202
181
160
139
-113
96

Protected

Template

Protected

Template

Protected

hAPO-2c

hAPO-3b

hAPO-3

bcl-W
bcl-XL
bcl-XS
bfl-1
bad
bik
bak
bax
bcl-2
mcl-1
-L32 113
GAPDH

401
364 (L)
324 (S)
286
257
226
202
181
160
139
--

Template

96

Caspase-8
FasL
Fas
Fadd
DR3
FAP
FAF
Trail
TNFRp55
Tradd
Rip
L32
GAPDH

406
353
316
283
253
226
202
181
160
149
134
113
96

Protected

Template

Protected

hAPO-3d
Caspase-8
FasL
Fas
DCR-1
DR3
DR5
DR4
Trail
DCR-2
TNFRp55
Tradd
Rip
L32
GAPDH

Template

Caspase-8
FasL
Fas
CLARP
FAP
CRADD
Daxx
MADD
RIP
--L32
GAPDH

406
351
316
253
226
199
163
151
134
--113
96

hAPO-4
406
351
316
286
254
227
202
181
170
160
149
134
113
96

Granzyme
Granzyme
Granzyme
Granzyme
Dad1
Fast K
RVP1
Dr-nm23
Requiem
CAS
Perforin
-L32
GAPDH

A
B
H
3

406
361
316
283
253
227
202
181
163
145
133
-113
96

www.bdbiosciences.com

Product Listing
Template

Protected

hAPO-5

Template

Protected

hAPO-5b

Template

Protected

hAPO-5c

XIAP
TRAF1
TRAF2
CART
NAIP
MIHC
MIHB
TRPM-2
CRAF
--L32
GAPDH

406
361
322
286
253
203
179
163
148
--113
96

TRAF1
TRAF2
CART
I-TRAF
TRAF5
TRAF6
CRAF
TRIP
---L32
GAPDH

361
322
286
253
226
163
148
136
---113
96

XIAP
survivin
NAIP
c-IAP-2
c-IAP-1
TRPM-2
-----L32
GAPDH

406
361
253
203
179
163
-----113
96

Template

Protected

Template

Protected

Template

Protected

hAPO-6
IPL
ASK1
Harakiri
SIAH
DFF
Nip2
Nip3
Nip1
DAP-K
DAP-K
DRM
L32
GAPDH

Template

Protected

mAPO-3
FLICE
FasL
Fas
FADD
FAP
FAF
TRAIL
TNFRp55L
TRADD
RIP
--L32
GAPDH

mAPO-2

mAPO-1
407
361
316
283
256
226
202
181
163
145
133
113
96

FLICE
YAMA
Mch2
Caspase 11
Caspase 12
ICH L/S
Mch3
ICE
Caspase X
caspase-2 (S)
--L32 112
GAPDH

406
325
283
253
226
202
181
163
151
137
---

Template

Protected

97

bclw
bclxL
bclxS
bfl1
bak
bax
bcl2
----bad
L32
GAPDH

406
364
272
239
202
182
160
----139
112
97

rAPO-1
406
353
316
283
226
202
181
160
149
134
--112
97

Fas Antigen
bcl-x(L)
bcl-x(S)
FasL
Caspase-1
Caspase-3
Caspase-2(L)
bax
bcl-2
Caspase-2(S)
--L32
GADPH

406
363(L)
325(S)
286
253
226
202(L)
181
160
137(S)
--113
97

Protected: Template size after RNAse digestion

www.bdbiosciences.com

2.1.1. Custom RiboQuant RNase Protection Assays

The RiboQuant RPA System


includes Multi-Probe Template Sets
designed to produce probes for a set
of biologically related genes. Over
100 Multi-Probe Template Sets have
been developed containing genes
arranged into groups according to
functional or structural relatedness.
In addition, to meet a variety of
research needs and the specifics of
individual experimental designs,
Pharmingen is providing Custom
RiboQuant services. These services
allow researchers to create unique
template sets by selecting DNA templates from any of the existing
Multi-Probe Template Sets or
requesting custom cloning of new
templates. Templates are available
for many different fields in cellular
and molecular biology including
immunology, neurobiology, cell cycle
regulation, DNA replication and
repair, apoptosis, development, and
gene regulation.

The Custom RiboQuant RPA


System provides the
following:
Custom Assembly:
Choose genes from existing inventory for the design of your own
template set from existing templates, the templates have to meet
two requirements:
1. Templates must come from the
same species - otherwise, aberrant
bands may be observed, which
impair proper analysis of the
results.
2. Templates with the same or
similar sizes cannot be used in one
template set. Usually, a size difference of appr. 10-15 bases is
required, preferrably more for
longer templates.
Custom Cloning:
If the gene(s) of interest is not in
our current inventory, we offer
custom cloning. We will design,
clone and generate templates to
place in a set customized for individual research needs. A prerequisite for this is that a sequence of
the corresponding gene has been
transmitted to Genebank.
Custom RPA Service:
Pharmingens Custom Technology
Team offers an RPA service that
will perform the analysis with
your RNA sample(s), using existing or customized Multi-Probe
Template Sets.

www.bdbiosciences.com

2.2. Atlas Arrays


Plastic or Nylon Macroarrays

High-quality, affordable
expression profiling
BD Biosciences Clontech offers a
complete platform for the analysis of
differential gene expression that
extends from RNA isolation to
bioinformatics.
Atlas Arrays, the central tool for
expression profiling, consist of
cDNA fragments or long oligos from
hundreds of genes immobilized on a
nylon membrane, plastic film, or
glass slide. Each gene included on an
Atlas Array is well characterized. By
focusing on known genes, Atlas
Arrays provide informative data
immediately. Although Atlas Arrays
provide sophisticated information,
the procedure for using them is
straightforward (Figure 1). The first
step is to prepare cDNA probes from
total or poly A+ RNA. You separately
hybridize probes to the Atlas Array,
perform a high-stringency wash, and
analyze the hybridization pattern.
Labeling and hybridization reagents
are provided.
The relative expression levels of each
gene can be assessed by comparing
the signals obtained from each array.
To automatically analyze Atlas
hybridization
results,
use
AtlasImage 2.01, a comprehensive
data analysis software package. For
visualizing expression patterns
across
multiple
samples,
AtlasNavigator provides a data
visualization and analysis tool to
help you focus on relevant genes. For
the highest quality radioactive
probes, we recommend using the
AtlasPure RNA Labeling System.
When using fluorescent probes, we
recommend using the Atlas Glass
Fluorescent Labeling Kit for uniform
label distribution.

Glass Microarrays

TM

TM

Atlas Glass
Fluorescent Labeling Kit (#K1037-1)

Atlas Pure Total RNA


Labeling Kit (#K1038-1)
TM

Atlas SMART
Probe Amplification Kit (#K1034-1)

Generate probes

TM

TM

Atlas Plastic
or Nylon Arrays

Atlas Glass
Microarrays

Profile gene expression

TM

AtlasImage

Fluorescent scanning
software

(#V1211-1)

Quantify raw array data

Analyze data to observe trends


TM

AtlasNavigator

(#V1220-1)

Obtain Atlas
gene information
TM

AtlasInfo

(free online database)

Profile expression
across tissues
Tissue expression profiling using
TM

Human RNA Chip


or
MTE Array (#7775-1)
or
Cancer Profiling Arrays (#7841-1, #7840-1)
TM

Figure 1. Atlas Array products take you from RNA isolation to localization of genes expression.

www.bdbiosciences.com

ments where it becomes necessary to


study highly homologous genes.
Whether using long oligos or cDNA
fragments,
our
bioinformatics
department always chooses the
region of the gene that has minimal
homology to any gene in the public
databases. This reduces cross
hybridization and gives you the
utmost confidence in each signal on
an Atlas Array.

Each gene on an Atlas Nylon Array


is represented by a cDNA fragment
(200600 bp). On our Plastic and
Glass Arrays, each gene is represented by a long oligo (80 bases). As
shown in Figure 2, cDNA fragments
have the highest hybridization efficiency, yet generally do not allow
discrimination between highly
homologous genes, like multigene
family members. These cDNA fragments are ideal for Atlas Nylon
Arrays that have a limited number of
genes (<1,176). Long oligos, on the
other hand, combine the high
hybridization efficiency of a cDNA
fragment with a short oligos ability
to distinguish homologous genes.
Long oligos are perfect for higher
density expression profiling experi-

Which array is right for you? It


depends on your needs. Nylon
Macroarrays are well suited for highquality expression profiling using a
limited gene set. These arrays allow
the use of 32P, and therefore, offer the
most sensitive measure of gene
expression (Table 1). In addition, no

special techniques or equipment is


needed to analyze Nylon Arrays.
Plastic Microarrays, like Nylon, do
not require special techniques or
equipment for analysis. Radioactive
detection is used for the most sensitive detection of gene expression.
Plastic has the added benefit of a
rigid and nonporous surface, which
facilitates high-throughput detection
with the lowest background possible.
Finally, Glass Microarrays permit
high resolution fluorescent detection
in a format that is best suited for
high-throughput analysis. See Table I
for a comparison of Plastic, Glass,
and Nylon Arrays. Whichever you
choose, Atlas Arrays give you access
to the entire public collection of
named human, mouse, or rat genes.

Table I: Comparison of Atlas Plastic, Glass, and Nylon


Plastic
8,000
Long oligos
33
P
+++
+++
Phosphorimager

# of Genes
Targets
Label/detection method
Relative sensitivity
Relative resolution
Imaging equipment
Multiple use
Analysis/ease of use

Yes
Easiest
No membrane deformation
++++
100% tested oligos
Yes

Homologous gene discrimination


Accuracy of spotted material
Calibration standards*

Glass
3,800
Long oligos
33
P
Fluorescence
+++
++
+++
++++
Phosphorimager
Fluorescent
scanner
No
Easiest
No slide deformation
++++
100% tested oligos
Coming soon

*For lot-to-lot comparison

Expression profiling
Oligo
array

Hybridization efficiency

cDNA
array

Homologous gene discrimination

Efficiency

Mutation
detection

0 10 20

100

200

600

1000

Oligos
cDNAs
Figure 2. The approximate length at which a DNA target achieves the optimal hybridization efficiency (yellow) and homologous gene discrimination (red).

www.bdbiosciences.com

Nylon
1,176
PCR-generated cDNA fragments
32
33
P
P
++++
+++
+
++
Phosphorimager or
autoradiogram
Yes
More difficult
Due to membrane deformation
++
100% sequence-verified
No

Atlas Human Apoptosis


Array

Atlas Hybridization and


Analysis Service

The Atlas Human Apoptosis Array


includes 205 genes that are known to
control apoptosis, including extracellular and cytoplasmic effectors:
Human Apoptosis Nylon Atlas
Array, 2 Membranes per Kit, 205
Genes, Cat. No. 7743-1

Let us perform an Atlas experiment


for you. Simply provide two or more
sampleseither frozen cells (>107) or
tissue (>50 mg) and indicate which
Atlas Nylon or Plastic Array to
probe. We isolate RNA from your
samples, generate and label cDNA
probes, hybridize the array, generate
phosphorimages, and analyze the
results using our AtlasImage software.

Many other Atlas Arrays contain


apoptosis-related genes, e.g.
Human Broad-coverage 1.2 Nylon
Atlas Array (Cat. No. 7850-1),
Human Cancer 1.2 Nylon Atlas
Array (Cat. No. 7851-1),
Mouse Toxicology 1.2 Nylon
Atlas Arrays (Cat. No. 7861-1),
Human 8K Plastic Microarray
(Cat. No. 7905-1),
Rat 4K Plastic Microarray (Cat.#
7909-1),
Human 7.6 Glass Microarrays
(Cat. No. 7912-1)

We deliver:
Phosphorimages of hybridized
membranes
AtlasImage software analysis output, indicating differential gene
expression
Follow-up technical support
For limited sample material we offer
Custom Atlas SMART Probe
Amplification.

Please visit our homepage


atlas.clontech.com

References for Atlas Arrays:


1. Yu-Wen E. Chang, Rolf Jakobi, Ann McGinty, Marco Foschi, Michael J. Dunn, and Andrey Sorokin. 2000:
Cyclooxygenase 2 Promotes Cell Survival by Stimulation of Dynein Light Chain Expression and Inhibition of
Neuronal Nitric Oxide Synthase Activity. Mol. Cell. Bio. 20: 8571 - 8579
2. Ali Khoshnan, Charles Tindell, Isett Laux, David Bae, Brydon Bennett, and Andre E. Nel. 2000: The NF-B
Cascade is important in Bcl-xL Expression and for the Anti-Apoptotic Effects of the CD28 Receptor in Primary
Human CD4+ Lymphocytes. J. Immunol. 165: 1743 - 1754
3. John D. Alvarez, Dag H. Yasui, Hiroyuki Niida, Tadashi Joh, Dennis Y. Loh, and Terumi Kohwi-Shigematsu.
2000: The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during
T-cell development. Genes & Dev. 14: 521 - 535
4. Warren M. Casey, Steven P. Anderson, Tony R. Fox, Dold M. Karen, Colton M. Heidi, and Morgan T. Kevin.
2001: Time course analysis relating the transcriptional and physiological responses of HepG2 cells exposed to
diethyl maleate. Physiol. Genomics. Dec 2001; 642001
5. Carl E. Clay, Genichi Atsumi, Kevin P. High, and Floyd H. Chilton. 2001. Early de Novo Gene Expression is
required for 15-Deoxy-12, 14-prostaglandin J2-induced Apoptosis in Breast Cancer Cells. J. Biol. Chem. 276:
47131 - 47135

10

www.bdbiosciences.com

2.3. HeLa & MCF7 Apoptosis cDNA Panels

Sets of cDNA for


Specifity
Clone Chimp
studying apoptosis
using quantitative PCR

during apoptosis found by array

RT-PCR allows you to determine the


in gene expression
between experimental samples over a
broader range1, 2. Because these panels are prepared from our Premium
RNA, which is virtually free of
genomic DNA, the chance of generating signals corresponding to
introns
or
other
noncoding
sequences is minimal. For optimal
PCR amplification, we recommend
our TITANIUM Taq DNA
Polymerase (Cat. No. 8434-1, -2).

with the
Baboon hybridization,
Cyno
Rhesus such
Pig as Sheep
Price ($)differences

Quickly reveal gene expression


during apoptosis
Span a range of apoptotic
conditions
Ready-for-PCR samples are
made with high-quality
Premium RNA
Includes two popular research
cell lines
Our Apoptosis cDNA Panels provide
a convenient way to quickly screen
for gene expression under different
apoptotic conditions. These panels
consist of cDNA samples synthesized
from cells treated with various apoptosis-inducing agents (Table I). These
samples allow you to use RT-PCR
and gene-specific primers to quickly
give a snapshot of that genes expression during apoptosis in different cell
model systems. In addition, the
cDNA samples can provide confirmation of differential expression

Atlas Apoptosis Array (Cat. No.


7743-1). Like our MTC (Multiple
Tissue cDNA) Panels, these panels
consist of sets of first-strand cDNA
carefully synthesized from total
RNA. We extract RNA for the HeLa
and MCF7 Apoptosis cDNA panels
from cells exposed to various wellknown apoptosis-inducing agents,
which span a range of mechanisms.
The HeLa panel contains first strand
cDNA from cells treated with either
the iron chelator desferrioxamine or
tumor necrosis factor (TNF).
The MCF7 Panel includes first
strand cDNA from cells expressing
wild-type, dominant-negative, or
p53 treated with or without doxorubicin. Doxorubicin is a DNA-damaging chemotherapeutic agent that
induces apoptosis through Myc.
The high sensitivity of RT-PCR lets
you detect rare mRNA transcripts
that might be missed by Northern
blotting or RNA dot blots, in only a
fraction of the time. Additionally,

Table I: Cell types and conditions for the Apoptosis cDNA Panels
HeLafor general apoptosis studies

MCF7to look at p53-related pathways

Control (untreated)

Wild-type control (untreated)

0.05 ng/ml TNF- for 2 hr

Wild type, 400 ng/ml doxorubicin for 7 hr

0.05 ng/ml TNF- for 5 hr

Wild type, 400 ng/ml doxorubicin 14 hr

250 M desferrioxamine for 6 hr

p53 dominant-negative vector control

250 M desferrioxamine for 20 hr

p53 dominant-negative vector, 400 ng/ml


doxorubicin treatment for 7 hr
p53 dominant-negative vector, 400 ng/ml
doxorubicin treatment for 14 hr

www.bdbiosciences.com

11

Quickly
across a
Body screen
Header
range of apoptotic
Body Sub-header
conditions

G3PDH
1

p21
1

Figure 1. Confirmation of up- or downregulated genes under different apoptotic conditions using the HeLa panel.
Panel A. G3PDH, a housekeeping gene.
Panel B. For the p21 gene, significant
up regulation was seen with 20-hr desferrioxamine treatment (lane 5), the
harshest of the conditions on the panels. Lane 1: control. Lane 2: TNF for 2
hr. Lane 3: TNF for 5 hr. Lane 4: desferrioxamine for 6 hr. Lane 5: desferrioxamine for 20 hr.

Having access to a single cell type


undergoing apoptosis via different
pathways gives you the ability to
quickly screen for activation of a
specific gene across a range of conditions that would be difficult to duplicate in the laboratory. If you were to
repeat this experiment yourself, you
would have to grow the cells, induce
apoptosis by various methods, wait
up to 20 hours, harvest the cells,
purify the RNA, and then use RTPCR to synthesize cDNA. The
Apoptosis cDNA Panels simplify
your work all you need are genespecific primers.
For focusing on p53-regulated apoptosis, we offer the MCF7 panel,
which contains unmodified MCF7
cells and MCF7 cells expressing
dominant-negative p53. The dominant-negative mutant contains
MDD2, a plasmid that expresses a
fragment of the p53 protein, and
that functionally inactivates p533, 4.
By comparing results obtained with
and without the p53 pathway, you
can see if the gene of interest is
induced in a p53-dependent fashion.
Once you have gene-specific primers,
you are ready to see if your target
gene is up or down regulated under
any of the conditions we offer.

Figure 1 shows two experiments


using the HeLa panel. It is well
known that NFB is induced when
cells are treated with TNF, and
that is supported by the panel NFB
expression is greater in the TNF
samples. The NFB experiment also
demonstrates that sometimes levels
of a gene increase when exposed to
apoptotic conditions, and then
decrease somewhat as gene expression slows down the progression of
apoptosis. We saw the increase in
binding of NFB to its DNA binding element by gel shift analysis
(data not shown). Unlike our MTC
Panels, the Apoptosis cDNA Panels
are not normalized. Each panel is
derived from a single cell line with a
set of specific treatments. Any differences seen are due to the treatmentthe total difference in time
between samples is a matter of
hours. If the samples were normalized, the apparent effects of the
treatment would be diminished. You
see no more than a two-fold variation in the expression level of
G3PDH, a housekeeping gene
(Figure 1A). For successful PCR
screening using the Apoptosis
cDNA Panels, you must have
enough sequence information to
design appropriate PCR primers,
and the PCR conditions should be
optimized for each new set of
primers. Included with each panel
are control DNA, detailed sample
source information, and a User
Manual.

Description

Size

Cat. No.

HeLa Apoptosis cDNA Panel


MCF7 Apoptosis cDNA Panel

10 rxns
10 rxns

K1440-1
K1441-1

References
1. Siebert PD, Huang BC. 1997: Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines. Proc. Natl.Acad. Sci. USA 94(6):21982203.
2. Human Multiple Tissue cDNA Panels (July 1997) CLONTECHniques XII(3):57.
3. Ossovskaya V.S, Mazo IA, Chernov MV, Chernova OB, Strezoska Z, Kondratov R, Stark GR, Chumakov PM, Gudkov AV.
1996. Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domain. Proc. Natl. Acad. Sci.
USA 93(19):1030910314.
4. Bacus SS, Yarden Y, Oren M, Chin DM, Lyass L, Zellnick CR, Kazarov A, Toyofuku W, Gray-Bablin J, Beerli RR, Hynes NE,
Nikiforov M, Haffner R, Gudkov A, Keyomarsi K. 1996. Neu differentiation factor (Heregulin) activates a p53-dependent
pathway in cancer cells. Oncogene 12(12):25352547.

12

www.bdbiosciences.com

BD Clontech

3. Protein Expression Analysis


3.1. Death Receptors and Ligands

Fas and Fas Ligand range of


apoptotic conditions

G ra nzy m e B

TRAIL

TRAIL

FasL

TNF

DR3

Fas

TNFR1

DR

ID

DAXX

FAD

8
s p ase

p ase 2

Ca

L ICE

Cas

p a s e 10
I- F

www.bdbiosciences.com

13

DD

FA P

ase 8

C a s p a s e 10

MA

FAD

C asp

TR A DD

IP

FA F

C as

Gr

an

zy m

eB

DR4
Per forin
Perforin

An area of particular interest has


been the induction of apoptosis by
the receptor-ligand pair: Fas (CD95)
and Fas Ligand (FasL). Fas is an
~42 kD cell surface protein which
belongs to the the TNF (tumor
necrosis factor receptor family).
FasL, an ~40 kD member of the TNF
ligand family, is expressed on activated T and NK cells. FasL initiates
signaling at the cell surface by aggregation of individual Fas receptors via
binding to the multivalent ligand.
Antibodies which selectively activate
Fas can be used in vitro to mimic the
apoptotic response associated with
FasL. Both Fas and FasL are thought
to play an important role in the
apoptotic processes that take place
during T cell development1, 2. BD
Pharmingen provides antibodies to
both human FasL and mouse FasL
which are suitable for analysis of
FasL expression by various techniques.

Per forin r forin


Pe

Apoptosis is one of a number of phenotypic responses that may occur as


a result of signal transduction pathways occurring in the cell. Clustering
of cellular receptors is a commonly
observed first step in the mechanism
of signal transduction pathways
which result in apoptosis. Clustered
receptor cytoplasmic domains trigger
subsequent steps in signal transduction pathways. An important link in
this system is provided by signal
molecules which bind directly to the
intracellular domains of receptors or
within a receptor signal complex.
Within the TNF receptor family
(TNFR types 1 and 2, Fas, TRAILR1 through -R4), many of these proteins fall into one of two categories
based on structural considerations:
the so called death domain homologues and the TNFR associated factors (TRAFs). These proteins share
sequence motifs which facilitate protein-protein interaction(s) with
receptors and other signal proteins
within the receptor complex. Thus,
these proteins provide an important
early step in the signal transduction
pathways that trigger apoptosis.

3.1. Death Receptors and Ligands (continued)

Untreated

Relative Cell Number

100
B

101

102

103

104

Treated with Ionomycin

TRAIL

TRADD

TRAIL-mediated pathways involve


activation of the transcription factor,
NFB, as well as activation of several members of the caspase family
of cysteine proteases. Several TRAIL
receptors have been identified, which
either activate TRAIL signal pathways, e.g., DR3, DR4 and DR5 or
may act as decoy receptors, socalled DcRs, e.g., DcR1, DcR2.

TRADD (TNF receptor associated


death domain) is a signal molecule
which interacts directly with the
cytoplasmic domain of TNFR
type I.6 Overexpression of the the Cterminal region of TRADD is sufficient to induce two major
TNFR-associated responses, apoptosis and activation of the nuclear transcription factor, NFB. CrmA
inhibits TRADD-induced apoptosis
but does not affect NFB activation,
suggesting that TRADD may serve to
initiate distinct signal pathways.
TRADD may interact with other signal proteins (FADD, TRAF1 and
TRAF2) as well as the serine/threonine kinase RIP.7

FADD

04

100

04

100

101

102

103

104

Treated with Ionomycin


+ KB8301

101

102

103

104

Flow cytometric analysis of Fas Ligand


(FasL) in peripheral blood mononuclear
cells (PBMCs). PBMCs were cultured for
3 hr in medium alone (A), Ionomycin
(B), or Ionomycin and a matrix metalloproteinase inhibitor, KB8301 (please
inquire). The cells were then stained for
flow cytometry with purified FasL antibody, clone NOK-1 (Cat. No. 556372),
followed by goat anti-mouse IgGbiotin. The cells were then incubated
with normal mouse serum before
adding anti-CD16-FITC (Cat. No.
555406) as a useful gate indicator and
Streptavidin-PE (Cat. No. 554061).

FADD (Fas associated death domain)


is a molecule involved in signal pathways mediated by members of the
TNF receptor family, including
TNFR type I, Fas and TRAIL-R1
(DR4).3,4FADD is a death domaincontaining adaptor protein that is
recruited to the receptor signal complex of activated cells. FADD may
interact with other signal proteins
(e.g., TRADD) or may bind caspases, including caspase-8 and caspase-10, thereby linking early
receptor events to an apoptotic protease cascade.5

RIP
RIP (receptor interacting protein) is a
serine/threonine kinase which may
be recruited to TNFR type I8 and
Fas9 receptor signal complexes in
activated cells. RIP may interact with
other signal proteins within these
complexes (e.g., RAIDD) and has
also been shown to interact with
pro-caspase-2.

References
1. Griffith, T.S. and T.A. Ferguson. 1997. The role of FasL-induced apoptosis in immune privilege. Immunol. Today 18:240244.
2. Lynch, D.H., F. Ramsdell and M.R. Alderson. 1995. Fas and FasL in the homeosta-tic regulation of immune responses.
Immunol. Today 16: 569-574.
3. Wajant, H., F. Johannes, E. Haas, K.Siemienski, R. Schwenzer, G. Schubert, T. Weiss, M. Grell and P. Scheurich.1998.
Dominant-negative FADD inhibits TNFR60, Fas/Apo1 and TRAIL-R/Apo2-mediated cell death but not gene induction. Curr. Biol.
8:113-116.
4. Yeh, W.C., J.L. Pompa, M.E. McCurrach, H.B. Shu, A.J.Elia, S. Shahinian, M. Ng., A. Wakeham, W. Khoo, K. Mitchell, W.S.
El-Diery, S.W. Lowe, D.V. Goeddel and T.W. Mak. 1998. FADD: essential for embryo development and signaling from some,
but not all, inducers of apoptosis. Science 279:1954-1958.
5. Muzio, M., A.M. Chinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Bretz, M. Zhang, R. Gentz,
M. Mann, P.H. Krammer, M.E. Peter and V.M. Dixit. 1996. FLICE, a novel FADD-homologue ICE/CED-3-like protease, is
recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85: 817-827.
6. Hsu, H., J. Xiong and D.V. Goeddel. 1995. The TNF receptor-1 associated protein TRADD signals cell death and NF- B activation. Cell 81:495-504.
7. Varfolomeev, E.E., M.P. Boldin., T.M. Goncharov and D. Wallach. 1996. A potential mechanism of cross-talk between the
p55 tumor necrosis factor receptor and Fas/Apo-1: proteins binding to the death domains of the two receptors also bind to
each other. J. Exp. Med. 183:1271-1275.
8. Ting, A.T., F.X. Pimentel-Muinos and B. Seed.1996. RIP mediates tumor necrosis factor receptor 1 activation of NF- B but
not Fas/APO-1-initiated apoptosis. EMBO J. 15:6189-6196.
9. Stanger, B.Z., P. Leder, T.H. Lee, E. Kim and B. Seed. 1995. RIP: a novel protein containing a death domain that interacts
with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 81:513-523.

14

www.bdbiosciences.com

TRAF3 and TRAF4

c-IAP-1

TRAFs (TNF receptor associated


factors) are signal transducing molecules which interact with members of
the TNF receptor family. Six TRAF
proteins have been identified to date.
TRAF1 and TRAF2 form a heterodimer that can interact with the
intracellular domain of TNFR type
II. TRAF2 also binds the intracellular region of CD3019, CD40 and a
novel member of the TNFR family,
ATAR.10 TRAF3 (CRAF1, LAP1)
was discovered based on its interaction with CD40 and the dominant
oncogene of Epstein-Barr virus,
LMP-1.11,12 TRAF4
(CART1), which binds human
CD40, has been implicated in breast
cancer.13 TRAF5, like TRAF2, has
been demonstrated to interact with
CD30,14 CD4015 and ATAR. TRAF6
interacts with the intracellular
domain of CD4015 and is unique
among the TRAF family proteins as
it has also been found to participate
in a signal pathway associated with a
receptor outside the TNFR family,
the IL-1 receptor.17

A family of inhibitors of apoptosis


(IAPs) have been identified which
can block apoptotic proteins, including certain caspases. The first human
IAP to be identified, NAIP, was discovered based on its association with
a neurodegenerative disorder. Other
IAPs include survivin, XIAP, c-IAP-1
and c-IAP-2. These proteins share
sequence motifs which promote protein-protein interaction(s) with caspases as well as with members of the
TRAF family of signal molecules.
IAPs display some specificity with
regard to their ability to bind and
inhibit caspases. Thus the IAPs may
have an important role in regulation
of apoptosis. c-IAP-1 migrates at ~72
kD on SDS-PAGE.18

10. Hsu, H., I. Solovyev, A. Colombero, R. Elliott, M. Kelley and W. J. Boyle. 1997. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5. J. Biol. Chem. 272:13471-13474
11. Cheng, G., A.M. Cleary, Z. Ye, D. Hong, S. Lederman and D. Baltimore. 1995. Involvement of CRAF1, a relative of TRAF,
in CD40 signaling. Science 267:1494-1498.
12. Mosialos, G., M. Birkenbach, R. Yalamanchili, T. VanArsdale, C. Ware and E. Kieff. 1995. The Epstein-Barr virus transforming protein LMP1 engages signaling pro-teins for the tumor necrosis factor receptor family. Cell 80:389-399.12.
13. Regnier, C.H., C. Tomasetto, C. Moog-Lutz, M.P. Chenard, C. Wendling, P. Basset and M.C. Rio. 1995. Presence of a new
conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is
expressed in breast carcinoma. J. Biol. Chem. 270:25715-25721.
14. Aizawa, S., H. Nakano, T. Ishida, R. Horie, M. Nagai, K. Ito, H. Yagita , K. Okumura, J. Inoue and T. Watanabe. 1997. Tumor
necrosis factor receptor-associated factor (TRAF) 5 and TRAF2 are involved in CD30-mediated NFkappaB activation. J. Biol.
Chem. 272:2042-2045.
15. Ishida, T.K., T. Tojo, T. Aoki, N. Kobayashi, T.Ohishi, T. Watanabe, T. Yamamoto and J. Inoue. 1996. TRAF5, a novel tumor
necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc. Natl. Acad. Sci. USA 93:9437-9442.
16. Ishida, T., S. Mizushima, S. Azuma, N. Kobayashi, T. Tojo, K.Suzuki, S. Aizawa, T. Watanabe, G. Mosialos, E. Kieff, T.
Yamamoto and J. Inoue. 1996. Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that
mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region. J. Biol. Chem. 271:28745-28748.
17. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama and D.V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature
383:443-446.
18. Rothe, M., M.B. Pan, W.J. Henzel, T.M. Ayres and D.V. Goeddel. 1995. The TNFR2-TRAF signaling complex contains two
novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83:1243-1252.
19. Tsitsikov, E.N., D.A. Wright and R.S. Geha. 1997: CD30 induction of human immunodeficiency virus gene
transcription is mediated by TRAF. Proc. Natl. Acad. Sci. USA 94:1390-1395.

www.bdbiosciences.com

15

kD
200
116
97
DR3

66
55
36
31
21

Western blot analysis of DR3, a receptor for the cytotoxic ligand TRAIL, in
Jurkat T cell lysate. Affinity purified,
rabbit anti-human DR3 (Cat. No.
556566) identifies DR3 as an ~ 59 kD
band.

Product Listing

Description

Clone(s)

Specificity

Daxx
DcR1
DR3
DR4
DR4
DR6
DRAK2
FADD
Fas

Polyclonal
Rabbit
Polyclonal
Polyclonal
Polyclonal
Rabbit
Rabbit
A66-2
DX2

Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu

Fas
Fas

G254-274
Jo2

Hu
Ms

NOK-1
NOK-2
G247-4
Kay-10

Hu
Hu
Hu
Ms

Fas Ligand
Granzyme B
Mcl-1
Perforin
Perforin Antibody Reagent Set
RIP
SODD
TRADD
TNF-

MFL3
2C5/F5
Polyclonal
dG9
dG9,27-35
G322-2
Rabbit
B36-2
MAb11

Ms

TNF-

MP6-XT22

Ms

TNF-

359-81-11

Hu

TRAF3
TRAF3
TRAIL
TRAIL
TRAIL

Polyclonal
B1-6
B35-1
RIK-1
RIK-2

Hu
Hu
Hu
Hu
Hu

Fas
Fas
Fas
Fas

Ligand
Ligand
Ligand
Ligand

Hu
Hu
Hu
Hu
Hu
Hu

Applications

Format

WB
WB
WB
WB
WB
WB
WB
IP, WB
FA, FC

Size

Cat. No. New Cat. No.

Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
NA/LE
Purified
FC
Biotin
FC
FITC
FC
PE
FC
CyChr
FC
APC
WB
Purified
IP, FC, FA
NA/LE
Purified
Biotin
FITC
PE
IP, FC, FA
NA/LE
IP, FA
NA/LE
IP, WB, FC
Purified
FC, FA
NA/LE
FC
Purified
FC
Biotin
FC
PE
FC
Purified
WB
Purified
IP, WB, IHCF, IHCP Serum
WB
Purified
FC
PE Set
IP, WB
Purified
WB
Purified
WB
Purified
FC
Purified
FC
FITC
FC
PE
FC
APC
FC
Purified
FC
FITC
FC
PE
FC
APC
FC, FA
NA/LE
FC
Purified
FC
PE
WB, IHCP
Serum
WB
Purified
WB
Purified
FC
Purified
FC
Purified
FC
Biotin
FC
PE

50 g
0.1 mg
50 g
50 g
50 g
50 g
200ul
0.1 mg
0.5 mg
0.1 mg
100 tests
100 tests
100 tests
100 tests
100 tests
0.1 mg
0.5 mg
0.5 mg
0.5 mg
0.5 mg
0.2 mg
0.25 mg
0.25 mg
0.1 mg
0.5 mg
0.5 mg
0.5 mg
0.2 mg
0.5 mg
50 g
0.1 ml
0.1 mg
100 tests
0.1 mg
200 l
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.5 mg
0.1 mg
0.1 mg
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.2 mg
0.1 mg

67051N
80056E
67061N
66901N
66891N
69481N
80446E
65751A
33450D
33451D
33452X
33454X
33455X
33458X
33459X
65311A
15400D
15401D
15402D
15404D
15405B
65320C
65330C
65431A
09930D
09931D
09932D
09935B
28101D
69901N
13656E
65991A
6599KK
65591A
80586E
66431A
18641A
18644A
18645A
18649A
18131A
18134A
18135A
18139A
18910D
18911A
18915A
66536E
66191A
66251A
69891A
69431A
69432B
69435A

556565
550622
556566
556544
556543
550439
550945
556402
555670
555671
555672
555673
555674
559773
558814
556370
554254
554255
554256
554257
554258
556371
556375
556387
553851
553852
553853
553854
555291
550558
554103
556434
556437
556395
550857
556496
554510
554512
554513
554514
554416
554418
554419
554420
554553
554554
554556
556506
556461
556468
550517
550515
550431
550516

IP, FC, FA
FA
FA
FA
E
E
E

100 g
10 g
10 g
10 g
20 x 96 tests
20 x 96 tests
20 x 96 tests

67231A
19761T
19321T
19771T
2649KI
2637KI
2673KI

556578
554618
554589
554619
555224
555212
555268

Related Reagents

Human Fas: FC Chimeric Fusion Protein Recombinant


TNF-
Recombinant
TNF-
Recombinant
TNF-
Recombinant
OptEIA Soluble Fas
OptEIA TNF-
OptEIA TNF-

16

www.bdbiosciences.com

Hu
Ms
Hu
Hu
Hu
Ms

Purified
Purified
Purified
Purified
Set
Set
Set

BD Pharmingen

3.2. Caspases

eB
zy m

an

APAF-

C a s p a s e 10
C as

C as

spa

me

p ase 4

p ase 8

C a s p ase 7

C a s p ase 3

C as

C a s p ase 9

p ase 1

C a s p ase 6

CrmA

C a s p ase 2

cI

XIA P
A P1 cIA P
SR EBP

A P -2 4

D 4 - G DI

D FF

L a m in s
PA RP

p17- 37
p17- 20
Pro-domain

p10-12

NH2

COOH

Pro-Caspases

Ca

C y to chro

se 8

Gr

Per forin

Per forin

Per forin

been grouped according to sequence


homology into three subclasses: The
caspase-1 subfamily (caspases-1, -4, 5, -11 and 12); the caspase-3 subfamily (caspases-3, -6, -7, -8, -9 and
-10) and caspase-2, which is the sole
member of the caspase-2 subfamily to date. Caspases are synthesized as inactive proenzymes (~30-55
kD) that are processed in cells undergoing apoptosis by self-proteolysis
and/or cleavage by another protein.
The processed forms consist of large
subunits (17-20 kD) and small (1012 kD) subunits which associate to
form an active enzyme. The large
subunits of these proteases also contain prodomain regions ranging from
as small as six amino acids to several
kilodaltons (kD). Therefore, the
molecular weight(s) of large subunits may be significantly larger due
to the presence or absence of the
prodomain, e.g., 35 kD for active
caspase-1. Functionally, active caspases form a proteolytic cascade,
capable of cleaving and activating
numerous cytoplasmic and nuclear
targets including PARP, D4-GDI,
DFF, MEKK and others.

Per forin

Cell death pathways are induced by


a variety of signals. One mechanism
which is consistently implicated in
apoptosis is the activation of a series
of cytosolic proteases, the caspases.
The term caspase reflects the catalytic properties of these enzymes,
the c denotes their cysteine protease mechanism and aspase refers
to their ability to cleave after aspartic acid residues. The caspase family
was originally discovered following a
search for mammalian homologs of
ced-3, a cell death gene described in
the nematode worm C. elegans. The
first mammalian caspase identified
was ICE (interleukin-1 converting
enzyme)1, now known as caspase-1.
Numerous other caspases have been
discovered and each has been given a
variety of names: caspase-1 (ICE),
caspase-2 (ICH-1, Nedd-2), caspase3 (CPP32, Yama, apopain), caspase4 (TX, ICH-2, ICErel-II, also
homologous to murine caspase-11),
caspase-5 (ICErel-III, TY), caspase-6
(Mch-2), caspase-7 (Mch-3, ICELAP3, CMH-1), caspase-8 (MACH,
FLICE, Mch-5), caspase-9 (Apaf-3,
ICE-LAP6, Mch-6), caspase-10
(Mch-4), caspase-13 (ERICE) and
caspase-14 (MICE). Caspases have

Nuclear Collapse
DNA fragmentation

Death

Activation by Cleavage
= Cleavage Site

Active Caspases

Activation of Caspases.
Caspases are synthesized as inactive proenzymes (pro-caspases) that are processed in cells undergoing apoptosis by
self-proteolysis and/or cleavage by another protease. The processed forms consist of large (17-20 kD) and small (1012 kD) subunits which associate to form an active enzyme.

www.bdbiosciences.com

17

WB IP/WB

3.2.1. Antibodies to Various


Caspases
Cleavage of caspases, which occurs
as part of the apoptotic pathway,
may often be demonstrated by western blotting techniques.
BD Biosciences Pharmingen has
developed and characterized monoclonal and polyclonal antibodies for
various caspases. These antibodies
are important tools in studying the
activation of caspases and their
roles in apoptosis using various
model systems.

116
97
66
55
Caspase-7

kD
116
97

kD

66

36
31

p17

14

Western blot analysis of caspase-3.


Jurkat T cells were untreated (0 hr, lane 1) or
treated (4 hr, lane 2) with camptothecin to induce
apoptosis. Cell lysates were then analyzed using
polyclonal caspase-3 antibodies (Cat. No. 556425).
The 17 kD caspase-3 cleavage product is seen in
the treated, but not in the untreated cell lysates.

kD
116
97
66
55

kD
116
97
66
55

p34

CrmA is a cowpox virus-encoded


protein that can protect cells
against apoptosis by inhibition of
caspases,2, 3, 4 with the highest affinity for caspase-1 and caspase-8.
Therefore, cells which express
CrmA may survive in the presence
of cytotoxic stimuli where these signal transduction pathways utilize
caspase-1 and -8. BD Pharmingen
provides a monoclonal antibody to
CrmA (Cat. No. 556427) which
may be used in western blotting to
determine the level of expression of
CrmA protein in cell and tissue
extracts.
kD
200

36
31

Caspase-6

116
97
66
55

21
14
p11

Western blot analysis of caspase-8.


Daudi B lymphoma cell lysates were probed with either
clone B9-2 (lane 1, Cat. No. 556466) or an isotype control(lane 2). The B9-2 antibody identifies caspase-8 as an
~55 kD band. The B9-2 antibody does not react with the
cleaved, active form of caspase-8.

p32

21

1 2 3 4 5

36
31

15
2

CrmA

14

Caspase-2

18
1

21

Western blot analysis of caspase-1.


Lysates from HeLa human cervical carcinoma cells (lane 1),
293 human embryonic kidney cells (lane 2), Jurkat T cells
(lane 3), BALB/c thymocytes (lane 4) and DC-3 SV40-transformed, rat ovarian granulosa cells (lane 5) were probed
with anti-caspase-1 (clone B24-2, Cat. No. 556497). The
antibody identifies the 45 kD (precursor) and 33 kD (intermediate) forms of human caspase-1. In mouse and rat
species, the antibody primarily detects the 45 kD form of
the protein.

29

Immunoprecipitation/western blot analysis of human


caspase-7.
Lane 1, 293 embryonic kidney cell lysate was probed
directly with clone B94-1 (Cat. No. 556541) which
detected caspase-7 at ~35 kD. Lanes 2 and 3, 293 cell
lysate was immunoprecipitated with clone B94-1 (lane 2)
or an isotype control (lane 3) and detected by western blot
analysis with B94-1. The bands above and below the specific band at ~35 kD represent the heavy and light chains
of IgG used for immunoprecipitation.

36
31

p33

44

36
31

Caspase-3

Caspase-1

67
Caspase-8

21

55

p45

kD

kD
200

6
1

CrmA
Western blot analysis of caspase-2.
Jurkat T cell lysates (lane 1), mouse myeloblasts (lane 2)
and DC-3 SV40-transformed, rat ovarian granulosa cells
(lane 3) were probed with anti-caspase-2 (clone G3101248, Cat. No. 554131). The G310-1248 antibody identifies caspase-2 (long form) as an ~48 kD band.

Western blot analysis of caspase-6.


Lanes 1 and 2, Daudi B cell lysate was probed with antihuman caspase-6 (clone B93-4, Cat. No. 556581, lane 1)
or with a mouse isotype control (lane 2). Clone B93-4 identifies full length caspase-6 as an ~34 kD band. Lane 3, purified, human caspase-6 (Cat. No. 556474),
which exists as a proteolytically cleaved dimer of 18 kD and
11 kD subunits, was probed with clone B93-4. The antibody identifies the 11 kD subunit of active caspase-6.

36
31
21
1

Western Blot Analysis of CrmA in Jurkat T Cells


Transfected with CrmA. Lane 1, anti-CrmA (clone A71-1,
Cat. No. 556427). Lane 2, a mouse IgG 1isotype control.

References
1. Cerretti, D.P., C.J. Kozlosky, B. Mosley, N. Nelson, K. Van Ness, T.A. Greenstreet, C.J. March, S.R. Kronheim, T. Druck, L.A. Cannizzaro, K. Heubner and R.A. Black. 1992. Molecular cloning
of the interleukin-1 converting enzyme. Science 256:97-100.
2. Komiyama, T., L.T. Quan and G.S. Salvesen. 1996. Inhibition of cysteine and serine proteinases by the cowpox virus serpin CrmA. Adv. Exper. Med. Biol. 389:173-176.
3. Tewari, M., L.T. Quan, K. ORourke, S. Desnoyers, Z. Zeng., D.R. Beidler, G.G. Poirier, G.S. Salvesen and V.M. Dixit. 1995. Yama/CPP32, a mammalian homolog of CED-3 is a CrmAinhibitable protease that cleaves the death substrate poly (ADP ribose) polymerase. Cell 81:801-809.
4. Zhou, Q., S. Snipas, K. Orth, M. Muzio, V.M. Vixit and G.S. Salvesen. 1997. Target protease specificity of the viral serpin CrmA. J. Biol. Chem. 272:7797-7800.

18

www.bdbiosciences.com

Product Listing

Description

Clone(s)

Specificity

Apaf-1
Apaf-1
Caspase-3, Active
Caspase-1
Caspase-10
Caspase-14
Caspase-14
Caspase-14
Caspase-2
Caspase-3
Caspase-3
Caspase-3
Caspase-3, Active
Caspase-3, Active
Caspase-3, Active
Caspase-3,
Active /FITC Mab Apoptosis Kit
Caspase-3,
Active Active- ELISA Ab Pair
Caspase-3, Active- ELISA Set
Active/PE MAb Apoptosis Kit
Caspase-3,
Caspase-4
Caspase-6
Caspase-7
Caspase-8
Caspase-8
Caspase-9
Caspase-9
Caspase-9
Caspase-9
c-IAP
Crm A
Cytochrome c
Cytochrome c
Hsp60
Hsp60
I-FLICE
Nedd4
Nedd4
Nedd4

24
24
C92-605
B24-2
Polyclonal
70A1426
32
32
G310-1248
Polyclonal
Polyclonal
Polyclonal
Polyclonal
C92-605
C92-605
C92-605

BD Pharmingen
BD Transduction Laboratories

Applications

Format

Size

Cat. No. New Cat. No.

Hu
Hu
Hu,Ms
Hu,Ms,R
Hu
Hu, Ms
Hu, Ms
Hu, Ms
Hu, Ms
Hu, Ms
Hu,Ms
Hu,Ms
Hu,Ms
Hu,Ms
Hu,Ms
Hu,Ms

WB, IF
WB, IF
FC, IF, IP
WB
WB
WB
WB, IF
WB, IF
WB
WB, IHCP
IHCF
FC
IP,IHCF,FC
FC
FC
FC

Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Biotin
PE
Purified
Biotin
PE
Kit

50 g
150 g
25 g
0.1 mg
50 g
150 g
50 g
150 g
0.1 mg
0.1 ml
0.1 mg
100 tests
0.1 mg
100 tests
100 tests
100 tests

A92820
68651G
68651G
66441A
67041N
80641F
C97020-50
C97020-150
13951A
65906E
67342A
67345X
67341A
68652X
68655X
6976KK

611364
611365
559565
556497
556564
550873
611510
611511
554131
556425
557038
557091
557035
550557
550821
550480

19 & C92-605

ELISA

ELISA

Pair

0,5 ml ea.

69941K

550578

19 & C92-605

ELISA

ELISA

Set

Set

8072KK

550930

C92-605
B25-1
B93-4
B94-1
B9-2
Rabbit
B40
Polyclonal
Rabbit
Rabbit
B75-1
A71-1
6H2.B4
7H8.2C12
24
24
Polyclonal
Rabbit
15
15

Hu, Ms
Hu,Ms,R
Hu
Hu
Hu
Hu, Ms
Hu
Hu
Hu
Hu
Hu
Viral Protein
Hu, Ms, R
Hu, Ms, R
Hu, Ms, R
Hu, Ms, R
Hu
Hu, Ms
Ms
Ms

FC
WB
WB
IP, WB
WB
WB
WB
WB
WB
WB
IP, WB
WB
IP
WB
WB
WB
WB
WB
WB, IF
WB, IF

Kit
Purified
Purified
Purified
Purified
Serum
Purified
Serum
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Purified
Purified

Kit
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0,1 ml
0.1 mg
0.1 ml
50 g
50 g
0.1 mg
0.1 mg
0.1 mg
0.1 mg
150 g
150 g
50 g
0,1 ml
50 g
150 g

8067KK
66171A
68041A
66871A
66231A
69236E
66571A
68086E
69461N
69471N
66791A
65921A
65971A
65981A
H99020-050
H99020-150
67071N
69976E
N95520-050
N95520-150

550914
556459
556581
556541
556466
559932
556510
556585
550437
550438
556533
556427
556432
556433
611562
611563
556567
550598
611480
611481

www.bdbiosciences.com

19

3.2.3. Antibodies to Caspase-3 Protein Substrates

Active caspase-3 can proteolytically


cleave numerous cellular targets in
apoptotic cells, including PARP, D4GDI and other molecules of inactive, pro-caspase- 3. During
apoptosis, PARP [poly (ADP ribose)
polymerase], an enzyme involved in
DNA repair and genomic maintenance, becomes proteolytically
processed from a 116 kD intact
form into 85 and 25 kD fragments.
BD Biosciences Pharmingen provides three antibodies to human
PARP: clones C2-10 (Cat. No.
556362), 4C10-5 (Cat. No.
556494) and 7D3-6 (Cat. No.
556493) which may be used to
identify the 116 kD form of PARP,
as well as the 85 kD cleaved fragment in cells which are actively
undergoing apoptosis. D4-GDI and
Rho-GDI are negative regulators of
Rho GTPases, proteins which are
thought to control the cytoskeletal
and membrane changes that accompany cell death. The mature 28 kD
form of D4-GDI can be cleaved by
caspase-1or caspase-3 into truncated
forms of 18 kD and 23 kD, respectively, a process which is believed to
play a role in regulation of Rho
GTPases during apoptosis. We provide polyclonal antibodies to human
Rho-GDI/D4-GDI (Cat. No.
556511) which can be used to identify the 28 kD form of D4-GDI, as
well as the 23 kD and 18 kD fragments in apoptotic cells. Other polyclonal antibodies to human D4-GDI
(Cat. No. 556498) may be used in
western blotting techniques to determine the level of expression of the
28 kD form of D4-GDI in cells.
Suggested references for cellular
proteins which are targets of caspase-mediated cleavage include:

20

www.bdbiosciences.com

D4-GDI
* Na, S., et al. 1996. D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis.
J. Biol. Chem. 271:11209-11213.
DFF
* Liu, X., et al. 1997. DFF, a heterodimeric protein that
functions downstream of caspase-3 to trigger DNA
fragmentation during apoptosis. Cell 89:175-184.
DNA-dependent protein kinase
* Casciola-Rosen, L., et al. 1996. Apopain/CPP32 cleaves
proteins that are essential for cellular repair: A fundamental principle of apoptotic cell death. J. Exp. Med.
183:1957-1964.
* Song, Q., et al. 1996. DNA-dependent protein kinase
catalytic subunit: A target for an ICE-like protease in
apoptosis. EMBO J. 15:3238-3246.
MEKK
* Cardone, M.H. et al. 1997. The regulation of anoikis:
MEKK-1 activation requires cleavage by caspases. Cell 90:
315-323.
MDM2
* Nicholson, D.W., et al. 1997. Caspases: killer proteases.
Trends Biochem. Sci. 22:299-306.
* Erhardt, P., et al.1997. Identification of the MDM2
oncoprotein as a substrate for CPP32-like apoptotic
proteases. J. Biol. Chem. 272:15049-15052.
PAK1/PAK2
* Walter, B.N., et al. 1998. Cleavage and activation of
p21-activated protein kinase gamma-PAK by CPP32
(caspase 3). Effects of autophosphorylation on activity. J.
Biol. Chem. 273:28733-28739.
PARP
* Nicholson, D.W., et al. Identification and inhibition of
the ICE/CED-3 protease necessary for mammalian
apoptosis. Nature 376:37-43.
* Tewari, M., et al. 1995. Yama/Cpp32b, a mammalian
homologue of CED-3, is a CrmA inhibitable protease
that cleaves the death substrate poly (ADP-ribose) polymerase. Cell 81:801-809.
SREBP-1,2
* Emoto, Y., et al. 1996. Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during
apoptosis. EMBO J. 15:1012-1020.
Rb
* Tan, X., et al. 1997. Degradation of retinoblastoma protein in tumor necrosis factor- and CD95-induced cell
death. J. Biol. Chem. 15:9613-9616.
For a more complete review of cellular proteins which are
targets of caspase-mediated cleavage, please see
Tan, X., et al. 1998. The caspase-RB connection in cell
death. Trends Cell Biol. 8:116-120.

Control

Staurosporine

Immunofluorescence Micrographs of
Cleaved PARP. In this experment, HeLa cells
were induced to undergo apoptosis with
staurosporine. The fluorescent micrographs
shown indicate that cleavage site-specific
PARP-FITC antibodies (polyclonal, Cat. No.
551528) stain the staurosporine-treated (B),
but not the control cells (A). A ' and B '
represent phase contrast correlates of A and
B respectively.

Product Listing

Description

Clone(s)

Specificity

D4-GDI
D4 GDI
Rho-GDI/D4-GDI
DFF (C-terminal)
DFF (C-terminal) Blocking Peptide
DFF (N-terminal)
DFF (N-terminal) Blocking Peptide
DNA-PK (p350)
DNA-PK (p350)
IL-1
PAK1/PAK2
PARP
PARP
PARP
PKC
Retinoblastoma Protein (Rb)
SREBP-1
SREBP-2

Polyclonal
97A1015
Polyclonal
Polyclonal

Hu
Hu, Ms
Hu
Hu

Polyclonal

Hu

Polyclonal
4F10C5
B122
Polyclonal
7D3-6
4C10-5
C2-10
MC5
G3-245
IgG-2A4
IgG-1C6

Hu,Ms,R
Hu,Ms,R
Ms
Hu
Hu,Ms,R
Hu
Hu,Ms
Hu,Ms,R
Hu,Ms,R
Hu
Hu

Applications

Format

Size

Cat. No. New Cat. No.

WB
WB
WB
WB
EBS
WB
EBS
IP,WB
IP,WB
FA
WB
IP,WB,FC
IP,WB,FC
WB
IP,WB,IHC
IP,WB,IHC
WB
WB

Serum
Purified
Serum
Purified
Peptide
Purified
Peptide
Serum
Purified
Purified
Serum
Purified
Purified
Ascites
Purified
Purified
Purified
Purified

0.1 ml
50 g
0.1 ml
50 g
0.1 mg
50 g
0.1 mg
0.1 ml
0.1 mg
0.25 mg
0.1 ml
0.1 mg
0.1 mg
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.1 mg

66456E
80521N
66586E
66921N
67281A
66911N
67291A
65586E
66141A
23410C
67416E
66391A
66401A
65196E
14871A
14001A
67351A
67361A

556498
550746
556511
556546
556995
556545
556996
556394
556456
557539
557053
556493
556494
556362
554207
554136
557036
557037

Western blot analysis of Caspase-3, D4-GDI and PARP during Fas-induced apoptosis. Jurkat
T cells were treated with anti-human Fas mAb, clone DX2 (Cat. No. 555670) and Protein G.
Cultures were harvested at various time points as indicated and analyzed by western blotting
using antibodies that recognize both the intact and cleaved forms of caspase-3 (polyclonal, Cat.
No. 556425), D4-GDI (polyclonal, Cat. No. 556511) and PARP (clone 4C10-5, Cat. No. 556494).
The intact forms of the proteins were detected at all time points; however, the cleaved forms
(caspase-3 [17 kD], D4-GDI [23 kD] and PARP [85 kD]) were only detected at 2 hr and beyond
after the induction of apoptosis.

BD Pharmingen

www.bdbiosciences.com

21

10 0

10 1

10 2

10 3

10 4

Fluorescence Intensity
Flow cytometric analysis of Bcl-2 in human
peripheral blood lymphocytes. Cells were stained
with Bcl-2/100 Antibody Reagent Set (Cat. No.
556357), which includes anti-human Bcl-2-FITC
(clone Bcl-2/100) and an IgG 1 -FITC isotype control.

kD
66
55
36
31
Bax

21
14
6
1

Western blot analysis of Bcl-X in K652 leukemia


cell lysate. Lane 1, clone 2H12 (Cat. No. 556499)
detects Bcl-X as an ~29 kD band. To demonstrate
specificity, the binding of clone 2H12 to the Bcl-X
protein was blocked by preincubation with 2H12
specific blocking peptide which is part of the BclX (2H12) Blocking Peptide Set (Cat. No. 556579)
prior to antibody probing steps (lane 2).

kD
200

Members of the Bcl-2 family play an


important role in regulating the
response of cells to a wide variety of
apoptotic signals. Bcl-2 is considered
to be novel among protooncogenes
because it blocks apoptosis in many
cell types, thus providing selective
survival advantage for cells and possibly contributing to tumorigenesis.
Other members of this family which
act as inhibitors of apoptosis include
Bcl-XL , Mcl-1 and A1. Several Bcl-2
family members also promote cell
death, e.g., Bad, Bak, Bax and
Bcl-XS . It is thought that proteinprotein interactions between Bcl-2
family members play an important
role in their function. For example,
Bax may form homodimers or heterodimers with either Bcl-2 and
Bcl-XL. Formation of Bax homodimers is thought to promote cell
death, whereas Bax heterodimerization with either Bcl-2 or Bcl-XL
appears to block cell death. Bad, a
pro-apoptotic Bcl-2 family member,
heterodimerizes with Bcl-2 and
Bcl-XL, but not with other Bcl-2
family members. When Bad heterodimerizes with Bcl-XL , it displaces Bax from Bax-Bcl-XL
heterodimers and promotes cell
death. More recently, members of
this family have been suggested to
play a role in ion channel formation
in vitro.

116
97
66
55
36
31

Bcl-X

21
1

Western blot analysis of Bax in Daudi B cell lysate.


Lane 1, clone 6A7 (Cat. No. 556467) detects Bax as
an ~21 kD band. To demonstrate specificity, the
binding of clone 6A7 to the Bax protein was
blocked by preincubation with 6A7 specific blocking peptide which is part of the Bax (6A7)
Blocking Peptide Set (Cat. No. 556573) prior to
antibody probing steps (lane 2).

22

www.bdbiosciences.com

Relative Cell Number

3.3. Bcl-2 Family Members

-3
4-3

f-1
Ra

Cytochro

bag

bad

bad

Cytochrome C

ba d
bcl-XL

bcl-XL
bax

AIF

Raf-1
ba d

bag

bcl-2

bcl-2

B I-1

bcl-2
bid

bcl-2
bax

bid
bax
bax

bax
bid

me

Product Listing

Description

Clone(s)

Specificity

Bad
Bad
Bak
Bak
Bak
Bax
Bax
Bax
Bax
Bax
Bid
Bid
Bid
Bcl-2
Bcl-2
Bcl-2 Antibody Reagent Set

P3F6
2G11
Polyclonal
G317-1
G317-2
6A7
G206-1276
Polyclonal
Polyclonal
4D2
Rabbit
7
7
Polyclonal
Bcl-2/100
Bcl-2/100, MOPC-21

Hu
Ms
Hu
Hu
Hu
Hu,
Hu,
Hu
Ms,
Ms
Hu,
Hu
Hu
Hu
Hu
Hu

Bcl-2
Bcl-2
Bcl-2 Antibody Reagent Set

4D7
6C8
6C8, Ha4/8

Hu
Hu
Hu

Bcl-2
Bcl-2 Antibody Reagent Set

Polyclonal
3F11, A19-3

Ms
Ms

Bcl-2
Bcl-w
Bcl-X
Bcl-X
Boo
Nip-1
Nip-1

Polyclonal
16H12
Polyclonal
2H12
D12-1932
5
5

Ms,
Hu,
Hu,
Hu,
Ms
Hu,
Hu,

Ms, R
Ms
R
Ms

R
Ms
Ms
Ms, R
R
R

Format

Applications

Size

Cat. No. New Cat. No.

Purified
Purified
Serum
Purified
Purified
Purified
Purified
Serum
Serum
Purified
Serum
Purified
Purified
Serum
Purified
FITC Set
PE Set
Purified
Purified
FITC Set
PE Set
Serum
FITC Set
PE Set
Serum
Purified
Serum
Purified
Purified
Purified
Purified

WB, IHC(P)
IP, WB
WB
WB, IHC(F)
WB
IP, WB
IP, WB, IHC(F), IHC(P)
IP, WB, IHC(F), IHC(P)
IP, WB, IHC(F), IHC(P)
IP
WB, IP
WB, IF
WB, IF
IP, WB, FC, IHC(F), IHC(P)
WB
FC
FC
IP, WB
IP, WB, FC, IHC(F)
FC
FC
IP, WB, IHC(F), IHC(P)
FC
FC
IP, WB, IHC(F), IHC(P)
WB
WB, IHC(F), IHC(P)
WB, IHC(P)
WB
WB, IF
WB, IF

0.1 mg
0.1 mg
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 ml
0.1 ml
0.1 mg
0.1 ml
50 g
150 g
0.1 ml
0.1 mg
100 tests
100 tests
0.1 mg
0.1 mg
100 tests
100 tests
0.1 ml
100 tests
100 tests
0.1 ml
50 g
0.1 ml
0.1 mg
50 g
50 g
150 g

66551A
13361A
66026E
65401A
65371A
66241A
13401A
13666E
13686E
13421A
69356E
697920-050
B97920-150
14371E
65111A
6511KK
6681KK
14831A
15131A
1513KK
6682KK
15616E
1502KK
6683KK
13456E
69911N
65186E
66461A
80611N
N79420-050
N79420-150

556508
554078
556440
556384
556382
556467
554082
554104
554106
554084
550365
611528
611529
554160
556354
556357
556535
554202
554231
554234
556536
554279
554221
556537
554087
550559
556361
556499
550860
611096
611097

EBS
EBS

Peptide Set
Peptide Set

0.1 mg ea
0.1 mg ea

67171K
68001K

556573
556579

Related Reagents

Bax (6A7) Blocking Peptide Set


Bcl-X (2H12) Blocking Peptide Set

BD Pharmingen
BD Transduction Laboratories

www.bdbiosciences.com

23

3.4. Cytochrome c

Cytochrome c is an electron transporting protein that resides within


the inter-membrane space of the
mitochondria where it plays a critical
role in the process of oxidative phosphorylation and production of cellular ATP.1 An increasing amount of
interest has been directed toward the
role which cytochrome c has been
demonstrated to play in apoptosis
processes. Following exposure to
apoptotic stimuli, cytochrome c is
rapidly released into the cytosol, an
event which may be required for the
completion of apoptosis in some systems.2 In the cytosol, or on the surface
of
the
mitochondria,
cytochrome c is bound by the protein
Apaf-1 (apoptotic protease activating factor2, 3), which also binds caspase-94 and dATP. Binding of

cytochrome c triggers activation of


caspase-9, which then accelerates
apoptosis by activating other caspases.
BD Biosciences provides two antibodies to cytochrome c which may
be used for immunoprecipitation
(Cat. No. 556432) or western blotting (Cat. No. 556433) from cell and
tissue extracts. Other reported applications for clone 6H2.B4 include
immunodepletion of cytochrome c
from cell extracts2 and immunolocalization studies utilizing fluorescence
microscopy.5, 6 Clone 7H8.2C12 has
also been published for immunolocalization studies utilizing fluorescence microscopy.7

References
1. Boyer, P.D., B. Chance, L. Ernster, P. Mitchell, E. Racker and E.C.Slater. 1997. Oxidative phosphorylation and photophosphorylation. Annu. Rev. Biochem. 46:955-1026.
2. Liu, X., C.N. Kim, J. Yang, R. Jemmerson and X. Wang.1996. Induction of apop-totic program in cell-free extracts:
requirement for dATP and cytochrome c. Cell 86:147-157.
3. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90(3):405-13.
4. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91(4):479-89.
5. Duckett, C.S., F. Li, Y. Wang, K.J. Tomaselli, Thompson, C.B. and R.C. Armstrong.1998. Human IAP-like protein regulates
programmed cell death downstream of Bcl-X L and cytochrome C. Mol. Cell Biol. 18:608-615.
6. Rosse, T., O. Reynald, L. Monney, M. Rager, S. Conus, I. Fellay, B. Jansen and C. Borner. 1988. Bcl-2 prolongs cell survival
after Bax-induced release of cytochrome c. Nature 391:496-499.
7. Yoshida, H., K. Young-Yun, R. Yoshida, A.J. Elia, A. Hakem, R. Hakem, J.M. Pen-ninger and T.W. Mak. 1998. Apaf1 is
required for mitochondrial pathways of apoptosis and brain development. Cell 94:739-750.

24

www.bdbiosciences.com

Product Listing

Description

Clone(s)

APAF-1
Apaf-1
Apaf-1
Caspase-9
Caspase-9
Caspase-9
Caspase-9
Cytochrome c
Cytochrome c

Polyclonal
24
24
B40
Polyclonal
Rabbit
Rabbit
6H2.B4
7H8.2C12

Specificity
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu, Ms, R
Hu, Ms, R

Applications

Format

Size

WB
WB, IF
WB, IF
WB
WB
WB
WB
IP
WB

Serum
Purified
Purified
Purified
Serum
Purified
Purified
Purified
Purified

0.1 ml
50 g
150 g
0.1 mg
0.1 ml
50 g
50 g
0.1 mg
0.1 mg

Cat. No. New Cat. No.


68076E
A92820-050
A92820-150
66571A
68086E
69461N
69471N
65971A
65981A

kD

kD
200

66
55

116
97
66
55
36
31
21
14
6

Cytochrome c
1

Immunoprecipitation/Western Blot Analysis of Cytochrome c. Clone


6H2.B4 (Cat. No. 556432) was used to immunoprecipitate cytochrome c
from P388D1 mouse lymphoma (lane 2), HeLa human carcinoma (lane
3), Jurkat T cells (lane 4), and NIH/3T3 mouse fibroblast (lane 5) cell
lysates. Cytochrome c was detected by western blot analysis using clone
7H8.2C12 (Cat. No. 556433) (lanes 1-5). Purified rat cytochrome c was
used as a protein standard in lane 1. Cytochrome c is detected at a
molecular weight of ~15 kD. The upper bands in lane 1 represent dimers
or multimers of the purified cytochrome c sample.

BD Pharmingen
BD Transduction Laboratories

556584
611364
611365
556510
556585
550437
550438
556432
556433

36
31
21
Cytochrome c

14
6
1

Western Blot Analysis of Cytochrome c in HL-60 Cells. Lane 1, clone


7H8.2C12 (Cat. No. 556433). Lane 2, a mouse IgG 2b isotype control. The
7H8.2C12 antibody identifies cytochrome c as an ~15 kD band.

www.bdbiosciences.com

25

3.5. Other Apoptosis-Related Proteins


3.5.1. Signal Transducers
kD
200
116
97
66
55
JNKK

36
31
21
1 2

Pa
M

Western blot analysis of JNKK in HeLa human carcinoma cell lysates. Lane 1, clone A32-1 (Cat. No
556400). Lane 2, mouse IgG 1 isotype control.
Clone A32-1 identifies JNKK as an ~45 kD band.

C as

p ase 3

JN

ac1

NIK

Growth
Factor

KKs

SK

20

1
as

K3

38

NF
ct k
iv e

K6

kD
200

Ik B
FkB

JN

Ik
k1
Ikk2 Ikk3

EKK

Apoptosis is one of a number of phenotypic responses that may occur as


a result of signal transduction pathways in the cell. Clustering of cellular receptors is a commonly observed
first step in the mechanism of signal
transduction pathways which result
in apoptosis. Clustered receptor
cytoplasmic domains trigger a variety of signal tranducing proteins.
These proteins are critical mediators
of apoptotic pathways beginning at
the cell surface, where they may participate directly within a receptor
signal complex, to the nucleus and
the activation of genes required during the apoptotic process.

af

From
TACI

EK

RK

116
97

KK

Cytoplasm
Jun

66
55

Atf2 TCF, ELK1


NFkB
NF-AT

36
31

Cell
Activation

21
1

Western blot analysis of IKK in Daudi B lymphoma cell lysate. Lane 1, anti-IKK , clone B78-1
(Cat. No 556532). Lane 2, a mouse lgG 2b isotype
control. Clone B78-1 identifies IKK as an ~85 kD
band.

kD
200
116
97

NIK

66
55
36
31
21
1

Western blot analysis of NIK in 293 embryonic


kidney cell lysate. Affinity purified, rabbit antihuman NIK (Cat. No. 556569) identifies NIK as an
~104 kD

26

www.bdbiosciences.com

Product Listing

Description

Clone(s)

Specificity

A20
ASK1
c-myc
IB
IB
IB
IKK
IKK
IKK
IKKB/IKK2
IKK/NEMO
IKK/NEMO
JNK1
JNK1
JNKK
JNKK
LITAF
LITAF
MEK1
MEK2
MEK2
MEKK1
NIK
N-myc
N-myc
PKR
PKR
PTEN
PTEN

E5-1619
Polyclonal
9E10
6A920
21
21
B78-1
Rabbit
Rabbit
10A9B6
54
54
G151-333
G151-666
G282-114
A32-1
30
30
Polyclonal
A7-1
Polyclonal
Polyclonal
Polyclonal
B8.4.B
N-Myc-2
23
23
2
2

Hu
Hu
Hu

BD Pharmingen
BD Transduction Laboratories

Hu,
Hu,
Hu
Hu
Hu
Hu,
Hu,
Hu,
Hu
Hu
Hu
Hu
Hu
Hu
Hu,
Hu,
Hu,
Hu,
Hu
Hu
Hu,
R
R
Hu,
Hu,

Ms, R
Ms, R

Ms, R
D, R
D, R

Ms
Ms
Ms
Ms

Ms

Ms, R
Ms, R

Applications

Format

Size

WB
WB
WB, IHC(P)
WB
WB, IF
WB, IF
IP/WB
WB
WB
WB
WB, IF
WB, IF
IP, WB, IVK
IP, WB
IP, WB
IP, WB
WB
WB
WB
WB
WB
WB
IP/WB
IP/WB
IP, WB
WB, IF
WB, IF
WB, IF
WB, IF

Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Purified
Serum
Serum
Purified
Purified
Purified
Purified
Purified
Purified
Purified

50 g
50 g
0.1mg
50 g
50 g
150 g
0.1 mg
50 g
50 g
50 g
50 g
150 g
0.1 mg
0.1 mg
0.1 mg
0.1 mg
50 g
150 g
0.1 ml
0.1 mg
0.1 ml
0.1 ml
50 g
0.1 mg
0.1 mg
50 g
150 g
50 g
150 g

Cat. No. New Cat. No.


80601N
68031N
14851A
80031N
I95020-050
I95020-150
66781A
69521N
69531N
80041N
I89920-050
I89920-150
15701A
15691A
13671A
65731A
L11520-050
L11520-150
13586E
65421A
13596E
66886E
67091N
66001A
14861A
P97220-050
P97220-150
P96520-050
P96520-150

550859
556580
554205
550620
611408
611409
556532
550444
550445
550621
611306
611307
554286
554285
554105
556400
611614
611615
554097
556386
554098
556542
556569
556438
554206
611514
611515
611500
611501

www.bdbiosciences.com

27

3.5.2. Tumor Suppressors

Tumor Suppressor genes normally


function to regulate and restrict cell
proliferation. Many tumor suppressors are components of intracellular
signaling pathways that enable a cell
to receive and process growthinhibitory signals from its surroundings. When a cell loses expression of
critical components of the signaling
network, it can lose its ability to
respond to certain extracellular
growth-inhibitory signals and proliferate. Growth deregulation resulting
from the absence of normal expression or function of a tumor suppressor gene can then lead to
uncontrolled cell proliferation and
tumor formation.
The p53 protein is critical to regulation of normal cell growth and is a
suppressor of tumor cell proliferation. Inactivation of p53 by a number of mechanisms, such as missense
mutations or interaction with oncogenic viral or cellular proteins, can
result in tumor progression.
Mutations and/or allelic loss of the
p53 gene is associated with a wide

variety of human tumors. Known to


have a role in transcriptional regulation, p53 suppresses various promoters containing TATA elements in an
apparently sequence-independent
fashion. p53 also binds to DNA in a
sequence-specific manner via recognition of a 20 bp consensus-binding
site. This interaction stimulates the
expression of genes downstream of
the p53 binding site. A number of
genes that contain p53-binding sites
have been identified, including
MDM2, GADD45, and muscle creatine kinase. It is thought that MDM2
is a feedback regulator of p53. In
addition, a p53-inducible gene, Cip1,
has been identified and shown to
suppress tumor cell growth in culture.
The retinoblastoma gene (Rb) product is well known as a tumor suppressor and is either absent or
mutated in many human tumors.
Retrovirus-mediated gene transfer of
the wild-type Rb gene into several
Rb mutant neoplastic cell lines suppresses their tumorgenicity. Rb is a

110kDa nuclear phosphoprotein that


undergoes differential phosphorylation during the cell cycle. During G1
phase, Rb is predominantly in a
hypophosphorylated
state.
It
becomes increasingly phosphorylated throughout the cell cycle until
late mitosis, when substantial
dephosphorylation
occurs.
Hypophosphorylated Rb interacts
with a number of cellular proteins
including the E2F transcription factor, several cyclins, RBP-1, RBP-2,
c-myc, N-myc, and p46. Rb is
thought to mediate its effects, in
part, via the repression of genes
required for proliferation. For example, Rb is specifically recruited to
promoters containing E2F sites and
actively represses E2F mediated transcription. Rb also stimulates the
activity of other transcription factors, although the mechanisms are
less clearly defined. Thus, Rb
appears to regulate transcription in
its aim to control cell growth.

Relative Cell Number

10 0

10 1

10 2

10 3

10 4

10 0

10 0

10 1

10 2

10 3

10 4

10 1

10

10 3

10 4

10 1

10

10 3

10 4

10 0

Fluorescence Intensity

Immunohistochemical staining of Rb in human osteosarcoma tissue sections.


Tissues were stained with a mouse IgG isotype control (upper left) or with clone G3-245
(upper right). To demonstrate specificity, clone G3-245 was preincubated with either a nonspecific (negative control) peptide (lower left) or with a G3-245 specific blocking peptide
(lower right). Arrows indicate positive staining with G3-245.

28

www.bdbiosciences.com

Flow cytometric analysis of Rb in permeabilized MOLT4 leukemia cells.


Cells were stained with a mouse IgG isotype control (A) or with clone G3-245, anti-Rb-PE
Antibody Reagent Set (Cat. No. 556539) (B). To demonstrate specificity, anti-Rb-PE was
preincubated with either a G3-245 nonspecific blocking peptide (C) or with a specific peptide (D)

Product Listing

Description

Clone(s)

Specificity

Applications

Format

Size

p33ING
p53
p53
p53

Polyclonal
G59-12
PAb 122
PAb 240

Hu,
Hu,
Hu,
Hu,

p53
p53
p53 Antibody Reagent Set

DO-1
DO-7
DO-7,27-35

Hu
Hu
Hu

WB, IF
IP, WB, IHC(F), IHC(P)
IP, WB, FC
IP, WB, IHC(F)
IP, WB,IHC(F)
IP, WB, IHC(F), IHC(P)
IP, WB, FC, IHC(F), IHC(P)
FC

p53

PAb 1801

Hu

IP, WB, IHC(F), IHC(P)

p53
Rb Antibody Reagent Set
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
-Amyloid (a.a. 1-40)
-Amyloid (a.a. 1-42)
-Amyloid (a.a. 1-43)

PAb 246
Ms
G3-245, MOPC-21 Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu

IP
FC
FC
IP, WB, IHC(F)
IP, WB, IHC(F)
IP, WB, IHC(F)
WB
IP, WB
IP, WB
IP, WB
IP, WB, IF
IP
IP
E, IHC(F)
E, IHC(F)
E, IHC(F)

Serum
Purified
Purified
Purified
Purified
Purified
Purified
FITC Set
Peptide Set
Purified
Purified
Purified
FITC Set
Peptide Set
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Serum
Serum

0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.25 mg
0.1 mg
0.1 mg
100 tests
100 tests
0.1 mg
0.25 mg
0.1 mg
100 tests
100 tests
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
25 g
25 g
25 g

66156E
14211A
14091A
14461A
14461C
15791A
15801A
1580KK
6680KK
14471A
14471C
14451A
6684KK
6685KK
14021A
14011A
14031A
14421A
14441A
14411A
14051A
14061A
14041A
14071A
66471G
66481G
66491G

556457
554157
554147
554166
554167
554293
554294
554298
556534
554169
554170
554165
556538
556539
554141
554140
554142
554163
554164
554162
554144
554145
554143
554146
556500
556501
556502

Hu
Hu
Hu

EBS
EBS
EBS
EBS
EBS
EBS

Peptide Set
Peptide Set
Peptide Set
Peptide
Peptide
Peptide

0.1 mg ea
0.1 mg ea
0.1 mg ea
250 g
250 g
250 g

67111K
67141K
66671K
66501C
66511C
66521C

556571
556572
556521
556503
556504
556505

G4-340
G3-349
C36
G99-73
G99-549
G99-2005
XZ55
XZ91
XZ104
XZ133
Polyclonal
Polyclonal
Polyclonal

Ms
Ms, R
Ms, R
Ms, R

Cat. No. New Cat. No.

Related Reagents

p53 (PAb 122) Blocking Peptide Set


p53 (DO-7) Blocking Peptide Set
Rb (G3-245) Blocking Peptide Set
-Amyloid (a.a. 1-40)
-Amyloid (a.a. 1-42)
-Amyloid (a.a. 1-43)

BD Pharmingen

www.bdbiosciences.com

29

3.6. Positive Control Lysates

BD Biosciences has recently


enhanced its already comprehensive
selection of cell and tissue lysates. An
even wider range of human, mouse,
and rat lysates are now available and
are ideal for assuring the integrity of
your western blotting experiments.
Now available for separate purchase,
these carefully prepared lysates are
Description

Jurkat
Jurkat
Jurkat
Jurkat
Jurkat

+
+
+
+
+

Staurosporine
Staurosporine
Staurosporine
Staurosporine
Staurosporine

Control Cell Lysate (0h)


Cell Lysate (1h)
Cell Lysate (4h)
Cell Lysate (8h)
Cell Lysate Kit

used for our in-house antibody


development and are optimized to
produce reliable western blot results.
We provide both treated and
untreated lysates for use as positive
controls for activity-dependent antibodies, such as our Apoptosis related
Antibodies.

Applications

Format

Size

Cat. No.

New Cat. No.

WB
WB
WB
WB
WB

Lysate
Lysate
Lysate
Lysate
Lysate

500 g
500 g
500 g
500 g
50 g each

611627
611628
611629
611630
611626

611627
611628
611629
611630
611626

3.7. Recombinant Proteins and Peptides

Description

Applications

Format

Size

Cat. No.

New Cat. No.

DFF (C-Terminal) Peptide


DFF (N-Terminal) Peptide
DR4 (C-Terminal) Peptide
DR4 (N-Terminal) Peptide
Fas (Soluble) Recombinant Protein (FasTM)
Fas : Fc Chimeric Fusion Protein
p53 (Wildtype) Recombinant Human Protein
p53 (Wildtype) Recombinant Human Protein

WB
WB
WB
WB
WB
IP
WB
WB

Purified
Purified
Purified
Purified
Purified
Purified
Lysate
Purified

100 g
100 g
100 g
100 g
10 g
100 g
0.5 ml
10 g

67281A
67291A
67261A
67271A
16341T
67231A
16226Y
66011T

556995
556996
556993
556994
554336
556578
554327
556439

30

www.bdbiosciences.com

BD Pharmingen
BD Transduction Laboratories

Apoptosis I

3.8. Protein Expression Screening


3.8.1. Antibody Sampler Kits

0h 0.5h 1h 4h 8h

0h 0.5h 1h 4h 8h

Screening
Analyze your experimental conditions with an array of antibodies to
identify which proteins to target for
further investigation.
Convenient
Each antibody is supplied in a 10 g
quantity and is ready for use.
Economical
Why pay much more to purchase
each antibody separately? Our
Antibody Sampler Kits allow you to
examine multiple proteins of interest
with minimal cost.

Format

Cat. No.

Kit

611427

Protein

Cat. No.

Apaf-1
Bad
Bax
Bcl-2
Bcl-x
BRUCE
CAS
hILP
Mcl-1
Nip1
p53

A92820
130
+
B36420
23kDa +
B73520
21kDa +
B46620
26kDa + nat/den
B61220
26kDa +
B84220 528kDa +
C42920 100kDa + nat/den
H59520 57kDa +
M54020 42/40kDa +
N79420 26kDa +
P21020
53kDa + nat/den

MW

WB

IP

IF IH
+
+
+
+
+
+
+
+

+
+
+

Apoptosis II

Reliable
All BD Transduction Laboratories
antibodies are developed in-house
and subjected to extensive screening
and quality control.
Controls
In addition to antibodies, each kit
contains the appropriate positive
control lysates for Western blotting at no additional charge!

0h 0.5h 1h 4h 8h

PARP
RIP
Caspase-2
Fas
DFF45
Fas Ligand
Caspase-7
TRADD
mCaspase-3
hCaspase-3
FADD

Antibody Sampler Kits in Action


In vertebrates, T cell development in
the thymus and development of the
nervous system are probably the best
characterized systems for the study
of programmed cell death. In the
experiment shown in fig. 1, human
Jurkat T cells were treated with the
apoptotic agent staurosporine, for
the indicated times. Cells were harvested, lysed, and analyzed by
Western blotting with the indicated
antibodies from the Apoptosis I and
Apoptosis II Antibody Sampler Kits.

Comprehensive
Each antibody kit targets both established and novel proteins within specific areas of research.
BRUCE
Apaf-1
CAS
hILP
p53
Mcl-1
Nip1
Bcl-x
Bcl-2
Bad
Bax

BD Transduction Laboratories
Antibody Sampler Kits provide a
unique way to examine your samples
for multiple proteins of interest without the costs of purchasing individual antibodies from a variety of
sources. Each kit is built around a
particular area of research and contains multiple antibodies. Since our
monoclonal antibodies are of the
highest quality and specificity, each
produces a distinct signal for its target protein. Each kit also includes
the appropriate positive control
lysates for Western blotting so you
can be assured of your results.

Format

Cat. No.

Kit

611428

Protein

Cat. No.

Caspase-2
Caspase-3
Caspase-3
Caspase-7
DFF45
FADD
Fas/CD95
Fas Ligand
PARP
RIP
TRADD

I75620
48kDa +
+
C31720
32kDa + nat/den +
C76920
32kDa +
M64620 35kDa +
D76320 45kDa +
F36620
24kDa +
+
F22120
45kDa +
+
F37720
37kDa +
den
+
P76420 113kDa +
+
R41220
74kDa + nat/den +
T50320
34kDa +
den
+

MW

WB

IP

IF IH

Each Antibody Sampler Kit includes 10 g of each


antibody listed at a concentration of 250 g/ml. No
substitutions allowed.
PARP
CAS

hILP

Caspase-7

RIP

{
DFF45

Nip 1

Caspase-2
Fig. 1 Jurkat cells were exposed to 4 M staurosporine for up to 8 hrs to induce apoptosis. A
variety of antibodies from our Apoptosis I and II Sampler Kits were used to probe Western blots
of apoptotic Jurkat cells.

www.bdbiosciences.com

31

3.8.2. BD PowerBlot Western Array Screening Services

BD Biosciences implemented a rapid


screening procedure that simultaneously measures changes of over 800
different proteins. This assay uses the
large library of powerful monoclonal
antibodies
developed
at
BD
Transduction Laboratories. Content
changes of fully characterized proteins are easily detected, measured
and stored by this procedure. The
multiple antibody approach will generate systematic data regarding the
levels, dynamics and distribution of
signal transduction proteins under
both normal and disease conditions.
We visualize the use of the multiple
antibody approach to catalog
changes of signal transduction proteins of normal cells and organisms,
and in particular how those proteins
change in response to drugs, xenobiotics and disease.

Features
A unique advancement in proteomics that measures changes in
protein expression
Complementary to current mRNA
expression screens such as the BD
Atlas Array or the BD
Riboscreen Membrane Array
Avoids the limitations of traditional protein screening techniques
such as 2-D Gel Electrophoresis
and Mass Spectrometry
Utilizes highly specific and sensitive monoclonal antibodies detecting sub-nanogram levels of protein
Carefully formulated antibody
combinations yield reproducible,
semi-quantifiable results
Immediate identification of proteins with altered expression
Reagents for follow-up studies are
well characterized and readily
available

BD PowerBlot in Action
Protein expression changes in SY5Y cells after exposure to NGF or staurosporine
Control

Lane 5

NGF

10

15

20

25

30

35

40

Control

Lane 5

Lane 5

10

15

20

25

30

35

40

15

20

25

30

35

40

Staurosporine

10

15

20

25

30

35

40

Lane 5

10

a AF6
182 kDa
b BM28
125 kDa
c PKC
78 kDa
d Janusin
180 kDa
e GDNFR
58 kDa
f Caspase-7
35 kDa
g PP1
36 kDa
h CarboxyPeptidase E
50 kDa
i Raft1
245/200 kDa
j N-Copine
62 kDa

Western blot images from Template A of the BD PowerBlot analysis. This template screens 183 of the 820 different antibody specificities. a thru j show various protein signals that significantly changed after exposure to NGF (100 ng/ml for 24 h) and/or staurosporine (500 nM for 16 h).

32

www.bdbiosciences.com

The Method
1. Lysed sample

2. SDS-PAGE

3. Transfer

Send us your control and treated


samples.

Submitted samples are loaded


for gel electrophoresis.

Proteins are transferred to


PVDF membranes.

6. Data analysis

5. Data capture

4. Antibody incubation

Images are subjected to automatic spot finding and spot


matching using PDQuest software (Bio-Rad).

Blots are run in triplicate.


Electronic images of blots are
captured using the Odyssey
Infrared Imaging System.

Membranes containing
samples are probed with
over 900 mAbs.

7. Data summary

8. Bioinformatics
A summary file (not shown) is provided which
lists all protein expression changes detected, in
order of confidence, 1 through 5, with 5 being
the highest confidence. The confidence level is
based on fold change, reproducibility, and signal intensity.
Level 5 - changes greater than 2-fold in triplicate from good quality signals.
Level 4 - changes 1.50-1.99-fold in triplicate
from good quality signals.
Level 3 - changes 1.25-1.49-fold in triplicate
from good quality signals.
Level 2 - changes greater than 1.25-fold in triplicate from low signals.

Data is exported to an electronic spreadsheet that indicates raw signal intensity data, normalized data,
and comparative analysis.

Level 1 - changes greater than 2-fold in duplicate from good quality signals.

Upon completion of the BD PowerBlot service, a results package is assembled for you, which includes the catalog numbers for all the antibodies used. If youre unsure
of the biological relevance of any of the proteins evaluated, a quick visit to our website (www.bdpowerblot.com) and a search by catalog number will yield a pdf file of
the technical document associated with your protein of interest.

www.bdbiosciences.com

33

3.8.3. Customize Your BD PowerBlot


Staurosporine-treated Jurkat cells

Know what you're looking


for?
Send us your samples, and we will
probe with antibodies you have
selected from our inventory.
We offer two Custom BD PowerBlot
formats:
1. Choose 40 individual antibodies
and we will test them all on a
single gel per lysate.
2. Choose 120 antibodies that we
will assemble into custom antibody cocktails for probing on a
single gel per lysate.
In addition to these two formats, we
can further customize BD PowerBlot
to fit your needs.

1 hr

4 hr

8 hr

Please contact your local BD


Biosciences office to get more information.
Fig. 1

Custom BD PowerBlot
Data
In this experiment, a customer used
the Custom BD PowerBlot screening
service to identify nuclear and/or
mitochondrial
markers
that
remained unchanged during staurosporine-induced apoptosis in
Jurkat cells. Control, 1-, 4-, and 8-hr
staurosporine-treated
lysates
(Jurkat+Staurosporine
apoptosis
lysate kit, Cat. No. 611626) were
loaded on gels and transferred to
PVDF membranes. A variety of anti-

bodies specific for housekeeping,


mitochondrial, and nuclear proteins
were used to probe the membranes.

Results
This Custom BD PowerBlot analysi
in fig. 1 shows decreases in the
expression of several proteins that
are degraded during apoptosis
(PARP, DFF45, Caspase-8, and
Caspase-7). Conversely, expression
of several proteins remained
unchanged, such as the RNA Pol. III
transcription termination factor La
Protein, the regulator of chromosome condensation RCC1, the mitochondrial enzyme Methyl-malonylCoA mutase, and the heat shock protein Hsp70. These may all be good
candidates for use as protein-loading
normalization controls in studies of
apoptosis. Interestingly, one of the
nuclear factors tested, TBP, was dramatically upregulated after staurosporine exposure. Thus, TBP may
play a key role in the transcriptional
regulation of genes involved in apoptosis.

BD PowerBlot Citations
1. Cunningham, B.A. 2001. Now Thats Service. The Scientist. 14(19). www.the-scientist.com.
2. Castedo M, Ferri KF, Blanco J, Roumier T, Larochette N, Barretina J, Amendola A, Nardacci R, Metivier D, Este JA, Piacentini
M, Kroemer G. 2001. Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J Exp Med. 194(8):1097110.
3. Arnoult D, Tatischeff I, Estaquier J, Girard M, Sureau F, Tissier JP, Grodet A, Dellinger M, Traincard F, Kahn A, Ameisen JC,
Petit PX. 2001. On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing
factor during Dictyostelium discoideum cell death. Mol Biol Cell. 12(10):3016-30.

34

www.bdbiosciences.com

4. Apoptosis Detection and Functional Analysis


Jurkat (human)

4.1. Antibodies to Active Caspase-3

bodies are useful for flow cytometric


analysis, immunohistochemistry of
acetone-fixed, frozen cytospins or
tissue sections and immunoprecipitation. The antibodies preferentially
recognize active caspase-3 as
opposed to pro-caspase- 3; although,
they may crossreact with pro-caspase-3 at high concentrations. Also,
please note that under denaturing
conditions, e.g., techniques such as
western blotting or immunohistochemistry involving formalin-fixation and/or paraffin-embedding,
antibody binding sites within active
caspase-3 may be exposed nonspecifically (not as a result of apoptotic
cleavage), therefore these antibodies
will react with both active and inactive, pro-caspase-3 using these methods.

Caspase-3 has been implicated as a


key protease that is activated during
the early stages of apoptosis.1, 2 Active
caspase-3, found in cells undergoing
apoptosis, consists of a heterodimer
of 17-21 and 12 kD subunits which
are derived from the 32 kD proenzyme. Active caspase-3 proteolytically cleaves and activates other
caspases, as well as relevant targets
in the cytoplasm and nucleus. BD
Pharmingens polyclonal and monoclonal antibodies recognizing the
active forms of human and mouse
caspase-3 are a new tool for identifying apoptotic populations. The antibodies were made against an active
human caspase-3 fragment and bind
to a conformational epitope which is
exposed by activation-induced cleavage or denaturation of the inactive
enzyme (pro-caspase-3). These antiJurkat
A

Untreated

99%
M1

95%
Relative Cell Number

Untreated

240

160

80

M1

120

5%
M2

+ Camptothecin

+ mouse Fas mAb

untreated

untreated

Immunohistochemical staining of apoptotic and nonapoptotic populations using anti-active caspase-3


antibodies. Jurkat T cells were treated for 4 hr with camptothecin to induce apoptosis (A) or left untreated (C). Mice
were treated for 6 hr with anti-mouse Fas mAb (clone Jo2,
Cat. No. 554254) to induce apoptosis (B) or left untreated
(D), and then sacrificed. Cytospins of Jurkat cells or frozen
tissue sections of mouse liver were acetone-fixed and
stained with biotinylated (A and C) or purified (B and D)
polyclonal, anti-active human caspase-3 antibodies [Cat.
Nos. 557038 (biotinylated) and 557035 (purified)]. The
Jurkat cells were then stained with an HRP-conjugated
Streptavidin second step, and the liver sections were
stained with a biotinylated goat anti-rabbit second step
(Cat. No. 550338) and then an HRP-conjugated
Streptavidin third step. Staining was visualized with a DAB
chromogen. The results show that active caspase-3 staining was identified only in cells or tissues induced to
undergo apoptosis.

kD

Mouse Thymocytes

kD

36
31

36
31

ProCaspase-3

1%
M2

Liver (mouse)

21
Active

14

21

Active
Caspase-3

14
6

6
C

Treated with
Camptothecin
(4 hr)

160
48%
80

M1

D
240
55%
M1

120

52%
M2

Treated with
mouse Fas mAb,
clone Jo2 (6 hr)
45%
M2

100

101

102

103

100

101

Immunoprecipitation;
pAb, Cat. No. 67341A
Western Blot;
pAb, Cat. No. 65906E

Western Blot;
pAb, Cat. No. 65906E

102

103

Log Fluorescence

Flow cytometric analysis of apoptotic and non-apoptotic populations using antiactive caspase-3 antibodies. Jurkat T cells (A, C) or mouse thymocytes (B, D) were left
untreated (A, B) or treated for 4 hr with camptothecin (C) or 6 hr with a mouse Fas
monoclonal antibody, clone Jo2 (Cat. No. 554254) (D) to induce apoptosis. Cells were
stained with PE-conjugated active caspase-3 antibodies (Cat. No. 557091). Untreated
cells were primarily negative for the presence of active-caspase-3, whereas about
half of the population of cells induced to undergo apoptosis had detectable caspase3 activity.

Caspase-3 Activation as a Marker of Apoptosis. In this experiment, proteins from


Jurkat T cell lysates were immunoprecipitated with monoclonal antibodies specific
for either the active form of caspase-3 [lanes 1 and 3: clone C92-605, Cat. No. 559565
(recognizes the 17-22 kDa large subunit of active caspase-3)] or for both procaspase3 (31 kDa) and active caspase-3 (17-22 kDa) (lanes 2 and 4: clone 19, Cat. No. 610323).
This was followed by western blot with polyclonal antibodies recognizing both pro
and active caspase-3 (Cat. No. 556425). The results show that active caspase-3 is present in campthothecin-treated cell lysates (lanes 3 and 4), but not in control cell
lysates (lanes 1 and 2), which contain procaspase-3 only (lane 2). Therefore, we
determine that only the camptothecin-treated lysate is apoptotic. Furthermore,
these data show that procaspase-3 is also present in the apoptotic population (lane
4). This indicates that not all cells in the population were undergoing measurable
apoptosis when the lysate was made. This is consistent with the dogma that cells in
a population do not generally undergo apoptosis at the same time.

References:
1. Nicholson, D.W., A. Ali, N.A. Thornberry, J.P. Vaillancourt, C.K. Ding, M. Gallant, Y. Gareau, P.R. Griffin, M. Labelle and M. Lazebnik. 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37-43.
2. Patel, T., G.J. Gores and S.H. Kaufmann. 1996. The role of proteases during apop-tosis. FASEB J. 10:587-597.

www.bdbiosciences.com

35

4.1.1. Monitoring Caspase-3 Activation during Apoptosis


using a novel ELISA System

Lysate Treatment
Camptothecin (4M)

1.2

Staurosporine (4M)
Control

1.0

We have developed an ELISA assay


based on the detection of active caspase-3, which enables high throughput screening for the presence of
Apoptosis (Figure 1). Assays performed on Jurkat T cells induced to
undergo apoptosis with camptothecin or staurosporine indicated
that active caspase-3 was detected in
lysates from treated cells but not
from control (untreated) cells (Figure
2).

Blank

OD 450nm

0.8

0.6

0.4

0.2

0
0.125

0.063

0.031

0.016

0.008

Active Caspase-3 Antibody (g/ml)

Figure 2. Active caspase-3 expression in camptothecin- and staurosporine-treated Jurkat cells.


Jurkat cells were left untreated (control) or
treated with 4 M camptothecin (4 hr) or 4 M
staurosporine (18 hr) to induce apoptosis. Active
caspase-3 was detected in apoptotic, but not in
control cells. The level of active caspase-3 in staurosporine-treated Jurkat lysate was 2.5 times
greater than in camptothecin- treated Jurkat
lysate. The capture antibody was used at 1.0 g/ml
and the detection antibody was serially diluted to
0.008 g/ml.

E
anti species
enzyme-labeled
antibody
detection
antibody

3.5

antigen

OD 450nm

3.0

capture
antibody

2.5
2.0

1.0
0.5
0.0
0

Treatment Time (hours)


Figure 3. Time course analysis of active caspase-3
levels following apoptosis induction. Jurkat cells
were left untreated (0 hr) or treated with 4 M
camptothecin to induce apoptosis. The level of
active caspase-3 increased progressively between
0 and 3 hr before reaching a plateau.

Staur (4M)
Control
Blank
Staur (4M)
Control
Blank

0.8

OD 450nm

1.5

Figure 1. Active caspase-3 ELISA Assay. The indirect


sandwich ELISA is based on the use of four components.
The first is a capture antibody (monoclonal mouse) that
detects pro- and active Caspase-3 (Cat. No. 610323); the
second is the antigen (cell lysate); the third is the detection
antibody that recognizes only the active form (Cat. No.
559565); and the fourth is the enzyme-labeled antisera
(goat anti-rabbit HRP, Cat. No. 554021) which is directed
against the detection antibody. The plates were developed
using TMB substrate kit (Cat. No. 555214) and were read
at 450 nm

Jurkat

Mcf-7

0.6

0.4

0.2

0
0.125

0.016

0.063

0.031

0.008

Active Caspase-3 Antibody (g/ml)

Figure 4. Analysis of active caspase-3 expression


in apoptotic Jurkat and MCF7 cells. Jurkat and
MCF7 cells were left untreated (control) or treated
with 4 M staurosporine (18 hr) to induce apoptosis. Active caspase-3 was detected in apoptotic
Jurkat cell lysates, but not in control cell lysates or
in apoptotic MCF7 lysates. The capture and detection antibodies were used as described in Figure 1.

36

www.bdbiosciences.com

More active caspase-3 was detected


in lysates from staurosporine- than
from camptothecin-treated cells,
indicating that different treatments
induce different levels of active caspase-3. We also showed, in a separate experiment, that the level of
active
caspase-3
progressively
increased over a three hour period
following camptothecin treatment
and then reached a plateau (Figure
3). As expected, active caspase-3 was
not detected in lysate from apoptotic
(staurosporine-treated) MCF7 breast
carcinoma cells (Figure 4), because
MCF7 cells lack caspase-3 expression due to a 47-base pair deletion
within exon 3 of the caspase-3 gene.
Taken together, the results indicate
that our new active caspase-3 ELISA
assay is specific for detecting active
caspase-3 in apoptotic cells.
This assay is useful for screening a
large number of samples for the presence of active caspase-3, and
expands our comprehensive offering
of state of the art reagents for studying apoptosis.

Product Listing

Description

Clone(s)

Specificity Applications Format

Size

Cat. No.

New Cat. No.

Fas
Caspase-3/CPP32
Caspase-3/CPP32
Caspase-3
Caspase-3, Active

Jo2
19
19
Polyclonal
Polyclonal

Ms

Caspase-3, Active

C92-605

Caspase-3, Active /FITC


mAb Apoptosis Kit
Caspase-3, Active/PE
mAb Apoptosis Kit
Caspase-3, Active/PE
pAb Apoptosis Kit
Caspase-3, Active
Active- ELISA Ab Pair
Caspase-3, Active- ELISA Set
Caspase-3/CPP32: HRPO
Caspase-3/CPP32: HRPO
Caspase-3/CPP32 antigen
Caspase-3/CPP32 antigen

C92-605

BD Pharmingen
BD Transduction Laboratories

Hu, Ms

IP,FC,FA
WB, IF
WB, IF
WB, IHC(P)
IP,IHC(F),FC
IHCF
FC
FC, IF, IP
FC
FC
FC
FC

NA/LE
Purified
Purified
Serum
Purified
Biotin
PE
Purified
Biotin
FITC
PE
Kit

0.5 mg
50 g
150 g
0.1 ml
0.1 mg
0.1 mg
100 tests
25 g
100 tests
100 tests
100 tests
100 tests

15400D
611320
611321
65906E
67341A
67342A
67345X
68651G
68652X
68654X
68655X
6976KK

554254
611320
611321
556425
557035
557038
557091
559565
550557
559341
550821
550480

C92-605

Hu, Ms

FC

Kit

100 tests

6865KK

550822

Polyclonal

Hu, Ms

FC

Kit

100 tests

6899KK

559762

19 & C92-605

Hu, Ms

ELISA

ELISA Pair

69941K

550578

19 & C92-605
19
19
46
46

Hu,
Hu,
Hu,
Hu,
Hu,

ELISA
ELISA
ELISA
ELISA
ELISA

ELISA Set
HRPO
HRPO
Rec. Prot.
Rec. Prot.

8072KK
C31725-050
C31725-150
C76920-050
C76920-150

550930
610324
610325
611048
611049

Hu, Ms

Ms
Ms
Ms
Ms
Ms

50 g
150 g
50 g
150 g

www.bdbiosciences.com

37

4.1.2. Human Active Caspase-3 Cytometric Bead Array (CBA)

The Human Active Caspase-3


Cytometric Bead Array (CBA) Kit is
a single bead assay for the rapid
semi-quantification of active caspase-3 in cell lysates by flow cytometry. The presence of active caspase-3
is an indication that cells are undergoing apoptosis. The Human Active
Caspase-3 CBA Kit combines the
principles of sandwich ELISA with
the capacity of using cell lysates in a
particle-based flow cytometric assay.
In this assay, cell lysates are incubated with particles (beads) coated
with antibodies that capture both
inactive and active caspase-3 proteins. The capture surfaces of the
beads are analogous to an individually antibody-coated well in an
ELISA plate. Fluorescently-labeled
reporter antibodies specific for active

caspase-3 are added to the bead mixture and the resulting reactions are
measured by fluorescent intensity
using a flow cytometer. The kit also
contains lyophilized apoptotic cell
lysate for generating active caspase-3
standard curves, which enables relative quantification of active caspase3 in the researchers cell lysate
samples. Heretofore, caspase-3 in
cell lysates has been typically analyzed by classical immunoprecipitation and western blot technologies.
These technologies are the most popular for analyzing intracellular proteins in cell lysates. Quantification is
generally restricted to such descriptors as detected or not detected, up
or down regulated, or fold changes
(e.g. two to ten fold). The Human
Active Caspase-3 CBA Kit offers an

alternative to both conventional


immunoprecipitation and western
blot assays. It is capable of measuring active caspase-3 over a two log
dynamic range using a simple 4 hr
assay protocol. The CBA technology
has comparable analytical sensitivity
and a wider dynamic range than conventional ELISA. With respect to
immunoprecipitation and western
blot techniques, CBA is more quantifiable, requires less sample, takes
less time, and is more amenable to
high throughput screening.

104
Wash

Control
Camptothecin

MFI

103

102

Analyze
10
Pro caspase-3
Active caspase-3
Other proteins
Capture mAb/Bead:
Pro/Active caspase-3

Detection mAb:
Active caspase-3PE

Requires less sample per assay


Achieve a wider dynamic range than ELISA
Save hands-on time
Reduce the total time to results

10

102

Size

Cat. No.

Human Active Caspase-3 CBA Kit


BD CBA Software (v1.1 and v1.2 are on the same CD-ROM)

100 tests
1 CD

552124
550065

www.bdbiosciences.com

104

P the
t iActive-Caspase-3
( )
Representative standard curve data from
CBA Kit. Standard
curves were generated from apoptotic and control Jurkat cell lysate. Jurkat cell cultures
were treated with 1 M camptothecin for 4 hr to induce apoptosis. Control cell cultures
were left untreated. Cell lysates were lyophilized, reconstituted, and serially diluted. The
data shows that active caspase-3 could be measured over a two log dynamic range in
apoptotic lysates. Control cells had minimal caspase-3 activity compared with apoptotic
cells.

Description

38

103

BD Pharmingen

4.2. Caspase Assays, Active Human Caspases, Substrates,


Inhibitors, Inducers and Sets
Caspase proteases play an essential
role in apoptosis by degrading specific structural, regulatory, and
DNA repair proteins within a cell.1, 2
Caspase-3 is an active cell-death
protease involved in the execution
phase of apoptosis, where cells
undergo morphological changes
such as DNA fragmentation, chromatin condensation, and apoptotic
body formation.3, 4
Caspase-3 is activated in response
to serum withdrawal, activation of
Fas, treatment with radiation, pharmacological agents4, as well as other
upstream caspases like caspase-8 and
caspase-9.
Caspase-8 is activated by the signaling pathways for CD95/Fas and
TNF, and is the most upstream caspase in the CD95 apoptotic pathway1. Upon activation, caspase-8
directly or indirectly initiates the
proteolytic activities of other caspases, including caspase-3.
Caspase-9 is activated very early in
the apoptotic cascade by the action
of cytochrome c, which is released
from the mitochondria in response
to apoptotic stimuli5. Activated caspase-9 then initiates the proteolytic
activity of other downstream caspases, including caspase-3 and caspase-6.

Caspases are activated by cleavage


at aspartate residues (D) resulting
from self-proteolysis and/or cleavage by another protease. The
processed forms consist of large and
small subunits which associate to
form active enzymes. When
expressed in E. coli, caspases spontaneously undergo autoprocessing
to yield the subunits characteristic
of the active enzymes. This system
has been used to produce active,
recombinant forms of human caspase-3, -6, -7 and -8, providing the
investigator with an additional tool
for characterization of apoptotic
pathways. The synthetic tetrapeptide substrates are proteolytically
cleaved by active human caspases.
The cleavage site is located between
D and AFC, AMC or pNA.
Released Fluorophores (AFC or
AMC) may be detected by ultraviolet (UV) spectrofluorometry.
Released chromophore pNA can be
monitored colorimetrically by
absorbance at 405nm.
When coupled to an aldehyde group
(CHO) or a fluoromethylketone
group (fmk), these tetrapeptides
function as potent inhibitors of caspase activity and can be used to
block caspase-mediated cleavage of
the corresponding fluorogenic substrates.

References
1. Cohen, G. M. 1997 Caspases: the executioners of apoptosis. Biochem J. 326:116.
2. Lazebnik Y A, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC. 1994: Cleavage of poly(ADP-ribose) Polymerase by
a proteinase with properties like ICE. Nature 371(6495):346-7.
3. Porter, A. G. & Janicke, R. U. 1999 Emerging roles of caspase-3 in apoptosis. Cell Death Diff. 6:99104.
4. Zou, H., Li, Y., Liu, X. & Wang, X. 1999 An APAF-1 cytochrome c multimeric complex is a functional apoptosome that
activates procaspase-9. J. Biol. Chem. 274:1154911556.
5. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahnad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-depedent
formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91(4):479-89.

www.bdbiosciences.com

39

DEVD-pNA

pNA

+ DEVD

Caspase-3
DEVD-AFC

IETD-pNA

AFC

pNA

+ DEVD

+ IETD

Caspase-8
IETD-AFC

AFC

+ IETD

4.2. Caspase Assays, Active Human Caspases, Substrates,


Inhibitors, Inducers and Sets (continued)
A

The Caspase Assay Kits and the


ApoAlert Caspase Assay Kits detect
caspase activation by assaying for
the cleavage of a fluorescent or
chemiluminescent substrate. The kits
allow to perform the assay directly
on cell lysates and are easily formatted for high-throughput applications.

Jurkat cells

Fluorescence units

2,500
2,000
1,500
1,000
500

+ Fas Ab
+ caspase-6 inh.

+ Fas Ab
+ caspase-9 inh.

+ Fas Ab

Fas Ab

NIH/3T3 cells

600

Caspase-3
+ Ac-DEVD-AFC

Caspase-6
+ Ac-VEID-AFC

Caspase-7
+ Ac-DEVD-AFC

A1

Caspase-3
+ Ac-DEVD-AFC
+ Ac-DEVD-CHO

B1

Caspase-6
+ Ac-VEID-AFC
+ Ac-VEID-CHO

C1

Caspase-7
+ Ac-DEVD-AFC
+ Ac-DEVD-CHO

800
600
400
200

+ Staurosporine
+ caspase-6 inh.

+ Staurosporine
+ caspase-9 inh.

+ Staurosporine

Staurosporine

Relative AFC Fluorescence

1,000

Fluorescence units

BD Biosciences Active Caspase Sets


includes the active caspase of interest, as well as a fluorogenic substrate
and inhibitor which are useful controls to test the activity of the
enzymes in your assays. These
reagents are suggested for use in
experiments in which the activity of
a specific caspase enzyme is desired.
In addition caspase fluorogenic substrates and inhibitors are available
separately.

400
200

0
600
400
200
0
450

Detection of caspase-9/6 activity in Jurkat and


NIH/3T3 cells. LEHD-AMC is cleaved by both caspase-9 and -6. Apoptosis was induced as indicated.
Cells were harvested and lysates were assayed as
described in the User Manual. Panel A. Jurkat cells
were incubated with or without Fas antibody (500
ng/ml) for 6 hr. Lysates were prepared and incubated with the caspase-9 substrate with or without caspase-9 or caspase-6 inhibitors. Panel B.
NIH/3T3 cells incubated with or without staurosporine (300 nM) overnight. Lysates were prepared and incubated with the caspase-9 substrate
either with or without caspase-9 or caspase-6
inhibitors.

480 500 520

550 450

480 500 520

550 450
(nm)

480 500 520

550

Activity of recombinant human caspases.


The synthetic tetrapeptide substrates Ac-DEVD-AFC, Ac-VEID-AFCand Ac-IETD-AFC are proteolytically cleaved by
active human caspases, releasing the fluorogenic molecule AFC, which may be detected by UV spectrofluorometry. When coupled to an aldehyde group (CHO), these tetrapeptides function as potent inhibitors of caspase activity and can be used to block caspase-mediated cleavage of these substrates. Active caspases-3, -6, -7 and -8 were
each incubated with a specific fluorogenic substrate alone (A-D) or in the presence of both substrate and inhibitor
(A1-D1). In the presence of active caspases, fluorogenic AFC is released from each substrate, demonstrating the
activity of the caspase enzymes. In the presence of both active caspases and caspase inhibitors, fluorogenic AFC
is not released, demonstrating that the activity of cas-pases -3, -6, -7 and -8 were blocked,

40

www.bdbiosciences.com

Product Listing
Description

Format/Conc.

Apps.

Size

Cat. No.

New Cat. No.

556485

Caspase-3

Caspase-3 Assay Kit


Ac-DEVD-AMC
Ac-DEVD-CHO
10 x PBS Buffer
Cell Lysis Buffer
2 x HEPES Buffer

Kit

SF

100 tests
5x200g
5x200g
50ml
50ml
50ml

6632KK

ApoAlert Caspase-3 Fluorescent Assay Kit

Kit

FA

25 assays
100 assays

K2026-1
K2026-2

ApoAlert Caspase-3 Colorimetric Assay Kit

Kit

FA

25 assays
100 assays

K2027-1
K2027-2

Active Caspase-3 Set


Active Caspase-3
Ac-DEVD-AFC
Ac-DEVD-CHO

Set

FA,SF

20 tests
5 g
200 g
20 g

6628KK

556473

556476

Cell Lysis Buffer, Reaction Buffer, DTT


DEVD-AFC Caspase Fluorescent Substrate
DEVD-CHO Caspase Inhibitor
Free Fluorophore

Cell Lysis Buffer, Reaction Buffer, DTT


DEVD-pNA Caspase Colorimetric Substrate
DEVD-fmk Caspase Inhibitor
Free Chromophore

Caspase-6

Active Caspase-6 Set


Active Caspase-6
Ac-VEID-AFC
Ac-VEID-CHO

Set

FA,SF

20 tests
5 g
200 g
20 g

6629KK

ApoAlert Caspase-9/6 Fluorescent Assay Kit

Kit

FA

25 assays
100 assays

K2015-1
K2015-2

Set

FA,SF

20 tests
5 g
200 g
20 g

6630KK

Kit

FA

25 assays
100 assays

K2028-1
K2028-2

Kit

FA

25 assays
200 assays

K2029-1
K2029-2

Set

FA,SF

20 tests
5 g
200 g
20 g

6631KK

Cell Lysis Buffer, Reaction Buffer, DTT, DMSO


LEHD-AMC Caspase 9/6 Fluorescent Substrate
LEVD-CHO Caspase-9 Inhibitor

Caspase-7

Active Caspase-7 Set


Active Caspase-7
Ac-DEVD-AFC
Ac-DEVD-CHO

556479

Caspase-8

ApoAlert Caspase-8 Fluorescent Assay Kit

ApoAlert Caspase-8 Colorimetric Assay Kit

Active Caspase-8 Set


Active Caspase-8
Ac-IETD-AFC
Ac-IETD-CHO

Cell Lysis Buffer, Reaction Buffer, DTT


IETD-AFC Caspase Fluorescent Substrate
IETD-fmk Caspase Inhibitor
Free Fluorophore

Cell Lysis Buffer, Reaction Buffer, DTT,


Dilution Buffer
Ac- IETD-pNA Caspase Colorimetric Substrate

Active Recombinant Caspases


Active Caspase-3
Ac-DEVD-AFC
Ac-DEVD-CHO

BD Pharmingen
BD Clontech

556482

5 g
200 g
20 g

www.bdbiosciences.com

41

Product Listing
Description

Format/Conc.

Apps.

Size

Cat. No.

Kit

FA

25 assays
100 assays

K2015-1
K2015-2

Purified, Active Recombinant Human Caspase-3

Enzyme

FA

Purified, Active Recombinant Human Caspase-6

Enzyme

FA

Purified, Active Recombinant Human Caspase-7

Enzyme

FA

Purified, Active Recombinant Human Caspase-8

Enzyme

FA

5 g
10 g
5 g
10 g
5 g
10 g
5 g
10 g

66281V
66281T
66291V
66291T
66301V
66301T
66311V
66311T

New Cat. No.

Caspase-8

ApoAlert Caspase-9/6 Fluorescent Assay Kit


Cell Lysis Buffer, Reaction Buffer, DTT, DMSO
LEHD-AMC Caspase 9/6 Fluorescent Substrate
LEVD-CHO Caspase-9 Inhibitor

Active Recombinant Caspases

556472
556471
556475
556474
556478
556477
556481
556480

Caspase Substrates and Inhibitors

Ac-VAD-fmk, Caspase Inhibitor


Ac-YVAD-AMC, Caspase-1 Fluorogenic Substrate
Ac-YVAD-fmk, Caspase-1 Inhibitor
Ac-DEVD-AFC, Caspase-3 Fluorogenic Substrate
Ac-DEVD-AMC, Caspase-3 Fluorogenic Substrate
Ac-DEVD-CHO, Caspase-3 Inhibitor
Ac-DEVD-CHO, Caspase-3 Inhibitor
Ac-DEVD-fmk, Caspase-3 Inhibitor
Ac-VEID-AFC, Caspase-6 Fluorogenic Substrate
Ac-VEID-CHO, Caspase-6 Inhibitor
Ac-IETD-AFC, Caspase-8 Fluorogenic Substrate
Ac-IETD-CHO, Caspase-8 Inhibitor
Ac-IETD-fmk, Caspase-8 Inhibitor

1 mM
SF
1 mM
SF
SF
SF
1 mM
1 mM
SF
SF
SF
SF
1 mM

100 l
1 mg
100 l
1 mg
1 mg
1 mg
100 l
100 l
1 mg
1 mg
1 mg
1 mg
100 l

66091U
67201U
66081U
66221U

66941U
66951U
66961U
66971U

8173-1
556451
8171-1
556574
556449
556465
8170-1
8172-1
556548
556550
556552
556554
8174-1

Apoptosis Inducer

Human TNF-

10 g

8157-1

AFC (7-amino-4-trifluoromethyl coumarin) emits a yellow-green fluorescence at 505 nm if excited at 400 nm after the substrate is cleaved by the protease.
AMC (7-amino-4-methoxy coumarin) fluorescence can be measured using a 380-nm excitation filter and a 460-nm emission, upon substrate cleavage.
Fluorescence detection is highly sensitive and can be used to measure even very small amounts of active caspase.
The caspase colorimetric assay measures the proteolytic cleavage of the chromophore p-nitroanilide (pNA) by caspases.
Liberated pNA can be monitored colorimetrically by absorbance at 405 nm.
When coupled to an aldehyde group (CHO) or fluoromethylketone (fmk), tetrapeptides function as potent inhibitors of caspase activity and can be used
to block caspase-mediated cleavage of the corresponding fluorogenic substrates.

ApoAlert Caspase Fluorescent Assay Kits were cited in the following articles:
Jingyu Diao, Aye Aye Khine , Farida Sarangi, Eric Hsu, Caterina Iorio, Lee Anne Tibbles, James R. Woodgett, Josef Penninger, and Christopher D. Richardson. 2001. X Protein of Hepatitis B
Virus Inhibits Fas-mediated Apoptosis and Is Associated with Up-regulation of the SAPK/JNK Pathway. J. Biol. Chem. 276(11):8328-8340
Saadi Ghatan, Stephen Larner, Yoshito Kinoshita, Michal Hetman, Leena Patel, Zhengui Xia, Richard J. Youle, and Richard S. Morrison. 2000. p38 MAP Kinase Mediates Bax Translocation
in Nitric Oxideinduced Apoptosis in Neurons. J. Cell Biol. 150(2):335347
Narvaez CJ, Welsh J.2001. Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells. J Biol Chem.;276(12):9101-7.
Delphine Lechardeur, Luke Drzymala, Manu Sharma, Danuta Zylka, Robert Kinach, Joanna Pacia, Christopher Hicks, Nawaid Usmani, Johanna M. Rommens, and Gergely L. Lukacs. 2000.
Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (ICAD/CAD) J. Cell Biol. 150(2):321334
ApoAlert Caspase Colorimetric Assay Kits were cited in the following articles:
Shasi V. Kalivendi, Srigiridhar Kotamraju, Hongtao Zhao, Joy Joseph, and B. Kalyanaraman. 2001. Doxorubicin-induced Apoptosis Is Associated with Increased Transcription of Endothelial
Nitric-oxide Synthase. J. Biol. Chem. 276(50):47266-47276
Yoshio Tomizawa, Yoshitaka Sekido, Masashi Kondo, Boning Gao, Jun Yokota, Joelle Roche, Harry Drabkin, Michael I. Lerman, Adi F. Gazdar, and John D. Minna. 2001. Inhibition of lung
cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B. Proc. Natl. Acad. Sci. +

42

www.bdbiosciences.com

BD Pharmingen
BD Clontech

4.2.1. Caspase3-Sensor Vector

In vivo Caspase activity


Monitoring Caspase-3 activity via
fluorescence microscopy
When using standard Caspase
assays, the results reflect the average
activity of Caspase-3 in the cells
lysed. The value can result from a
very high activity in a small part of
these cells, or from a pretty low
activtiy in all of them. With our new
BD Clontech pCaspase3-Sensor
Vector it is now possible to analyze
Caspase activity on a cellular level by
looking at the distribution of the fluorescent Caspase3-Sensor protein.
This vector encodes the enhanced
yellow-green variant (EYFP) of the
Aequorea victoria green fluorescent
protein (GFP) fused at the 3' end to
three copies of the nuclear localization signal (NLS) of the Simian Virus
40 large T-antigen. At the 5' end the
gene contains a sequence encoding
the nuclear export signal (NES) of
the Map Kinase Kinase (MAPKK).
The NES is separated from the EYFP
coding region by a 36-nucleotide
sequence encoding the region of Poly
(ADP-ribose) polymerase (PARP)
cleaved by caspase-3. The complete
coding sequence for this fusion protein is human codon-optimized. The
fluorescence excitation maximum of
EYFP is 513 nm; the emission spectrum has a peak at 527 nm and the
fluorescence intensity is roughly
equivalent to that of EGFP.

EYFP NLS

detect the onset of caspase-3 activity


in mammalian cells. This vector
allows visual monitoring of
caspase-3 activity in the cell via fluorescence microscopy. Because the
NES of MAPKK dominates the NLS,
the full-length fluorescent fusion
protein distributes to the cytosol.
Therefore a cytosolic fluorescent signal indicates that caspase-3 is not
active. However, if caspase-3 is activated, the dominant nuclear export
signal (NES) will be cleaved from the
fusion protein and the truncated fluorescent fusion protein will translocate to the nucleus via the NLS. The
translocation of the fluorescent protein from the cytosol to the nucleus
indicates caspase-3 activity
(Figure 1).
Now it is possible to analyze
Caspase-3 activity in vivo on a cell to
cell level. If desired, stable transfectants can be selected using G418.
pCaspase3-Sensor assays are very
sensitive and allow diverse experimental approaches. For example the
Caspase3-Sensor can be coexpressed
with a protein that might inhibit caspase activity at a specific point of the
cell cycle. For studying inhibitory
proteins or peptides, a coexpression
of the Caspase3-Sensor and the
potentially inhibitory protein fused
to DsRed might be performed. The
Caspase3-Sensor allows the analysis
of the Caspase-3 pathway with all
kinds
of
proteins,
peptides,
inhibitors and induction methods in
specific subpopulations of cells.

NES DEVD EYFP NLS


+ Stsp (h/4h)

NES DEVD

Nucleus

Figure 1.

pCaspase3-Sensor can be used to

Description

Size

Cat. No.

pCaspase3-Sensor Vector

20 g

8185-1

BD Clontech

www.bdbiosciences.com

43

EYFP NLS

4.3. ApoAlert Bid Vectors: DsRed2 & EGFP

New cell-based reporters


for monitoring a Bcl-2
family protein - Bid
Monitor Bid translocation in living cells
Identify and map a proteins proor anti-apoptotic properties
Evaluate agents for their effects on
Bid-dependent apoptotic pathways
Perform multicolor analyses
Now you can investigate apoptosis
in Living Colors. Our BD
Clontech ApoAlert pDsRed2-Bid
and pd4EGFP-Bid Vectors encode
biologically active fluorescent fusion
proteins that respond to any stimulus affecting the subcellular distribution of Bid a 22kDa, pro-apoptotic
protein known to play a vital role in
pathways that co-opt mitochondria
for apoptosis1. Each vector encodes
Bid fused with a Living Colors
Fluorescent Protein: red fluorescent
protein, DsRed22, or destabilized
enhanced green fluorescent protein,
d4EGFP3. Because of its fluorescent
label, the Bid fusion is easily detected
by microscopy, allowing you to track
the protein as it translocates from
the cytosol to the mitochondria in
response to certain apoptotic stimuli.
You can use pDsRed2-Bid or
pd4EGFP-Bid to study the suppression and induction of Bid translocation at all stages of apoptosis in
many types of mammalian cells.

translocation is triggered by caspase8, which, when activated by a death


signal, cleaves Bid to produce a
15kDa C-terminal fragment. After
being myristoylated5, the fragment,
often referred to as truncated Bid or
tBid, transmits the death signal by
selectively binding the outer mitochondrial membrane6.

A ready-to-use fluorescent
reporter
By transfecting cells with pDsRed2Bid or pd4EGFP-Bid, you can easily
detect this translocation using conventional fluorescence microscopy
(Figure 1)*. And because you can
examine individual cells, you can
design cotransfection and multicolor
experiments to study the effects
other proteins have on this crucial
apoptotic event. Once you identify a
proteins pro- or anti-apoptotic
property, you can go on to map the
domains responsible for inducing or
blocking the Bid-dependent pathway.
Additionally, by visualizing Bid in
conjunction with appropriate cellcycle indicators, you can investigate
the potential cell cycle dependence of
Bid translocation. Assays that detect
apoptosis in bulk populations cannot
provide this information. Now you
can dissect the Bid-dependent pathway in an easy non-invasive manner.

The role of Bid in apoptosis


The translocation of Bid is a widely
studied event in apoptosis. In
healthy, non-apoptotic cells, Bid, a
member of the Bcl-2 protein family,
usually resides in the cytosol. But
soon after the induction of apoptosis, it translocates to mitochondria,
where it facilitates the release of
cytochrome c a key amplification
step in the apoptotic cascade4. The

44

www.bdbiosciences.com

* Which vector you use may depend on whether you plan


to co-express other fluorescently labeled proteins as part of
a two- or three-color analysis. With the pd4EGFP-Bid
Vector, d4EGFP serves not only as a label, but also as a
device to ensure protein turnover. With a half-life of about
four hours, d4EGFP targets the Bid fusion for rapid degradation, ensuring that Bid-d4EGFP expression is maintained
at a low, but detectable level.

Untreated

Treated

Figure 1. Monitoring Bid activation with the pDsRed2- Bid and pd4EGFP-Bid
Vectors. 3T3 cells were transiently transfected with either pDsRed2-Bid (Panels A & B) or
pd4EGFP-Bid (Panels C & D). Approximately 24 hr later, cells were incubated with 700 nM
staurosporine, an apoptosis inducing agent, at 37C for 3 hr. Cells were then fixed and
examined with a Zeiss Axioskop equipped with the appropriate light filters. In untreated
cells (Panels A & C), the Bid reporters (Bid-DsRed2 and Bid-d4EGFP) distribute uniformly in
the cytosol, as expected. Following induction (Panels B & D), the reporters translocate to
the surface of mitochondria, forming intensely fluorescent clustersclear evidence that
the Bid pathway has been activated.

Description

Size

Cat. No.

pDsRed2-Bid Vector
pd4EGFP-Bid Vector

20 g
20 g

6977-1
6979-1

Notice to purchaser for Living Colors Products


Use of BD Biosciences Clontechs Living Colors products
contain-ing DNA sequences coding for mutant Aequorea
victoria green fluo-rescent protein (GFP) variants or proteins thereof requires a license from Aurora Biosciences
Corporation under U.S. Patent Nos. 5,625,048; 5,777,079;
6,054,321 and other pending U.S. and foreign patent
applications. In addition, certain BD Biosciences Clontech
products are made under U.S. Patent No. 5,804,387
licensed from Stanford University. Not-For-Profit research
institutes or entities are granted an automat-ic license
with the purchase of this product for use in non-commercial internal research purposes, the terms of which are
disclosed in detail in the license that accompanies the
shipment of this product. Such license specifically
excludes the right to sell or otherwise transfer this product or its components to third parties. For-Profit research
institutes or entities must obtain a license from Aurora
Biosciences Corporation. Please contact your local representative or www.aurorabio.com. Please contact BD
Biosciences Clontech directly for any other assistance,
including purchasing and technical support. All companies and institutions purchasing Living Colors products
will be included in a quarterly report to Aurora
Biosciences Corporation, as required by the
Clontech/Aurora license agreement.

References
1. Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH. 2000. Pro-apoptotic cascade activates BID, which
oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 7(12):1166-73. Review.
2. Living Colors DsRed2 (July 2001) CLONTECHniques XVI(3):2-3
3. Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C, Huang CC, Kain SR. 1998. Generation of destabilized green fluorescent
protein as a transcription reporter. J Biol Chem 273(52):34970-5.
4. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC.1999.
Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell
Biol.144(5):891-901.
5. Zha J, Weiler S, Oh KJ, Wei MC, Korsmeyer SJ. 2000. Posttranslational N-myristoylation of BID as a molecular switch for
targeting mitochondria and apoptosis. Science. 290(5497):1761-5.
6. Lutter M, Fang M, Luo X, Nishijima M, Xie X, Wang X. 2000. Cardiolipin provides specificity for targeting of tBid to mitochondria. Nat Cell Biol. 2(10):754-61.

BD Clontech

www.bdbiosciences.com

45

Notice to Purchaser for DsRed and its variants


This product is the subject of pending U.S. patents. NotFor-Profit-Entities: Orders may be placed in the normal
manner by contacting your local representative. BD
Biosciences Clontech grants not-for-profit research entities a worldwide, non-exclusive, royalty-free, limited
license to use this product for non-commercial life science
research use only. Such license specifically excludes the
right to sell or otherwise transfer this product or its components to third parties. Any other use of this product
will require a license from BD Biosciences, Clontech.
Please contact your local representative For-Profit entities
that wish to use this product in non-commercial or commercial applications are required to obtain a license from
BD Biosciences Clontech. For license information, please
contact your local representative.

4.4. ApoAlert Glutathione


Detection Kit

Fluorescence units

3,500
3,000
2,500
Control
1 min UV
5 min UV
15 min UV

2,000
1,500

Induce
apoptosis

1,000
500
0
0

Centrifuge & collect pellet

Time after induction (hr)

1 M Staurosporine

Wash pellet to remove


all traces of medium

3,500

Fluorescence units

BD
Biosciences
Clontech
ApoAlert Glutathione Detection
Kit is a quantitative in vitro assay
that detects decreased cytosolic glutathione (GSH) levels that occur
early in apoptosis in some cell types.
In healthy cells, glutathione acts as a
redox buffer. However, in Jurkat and
some other cell types, the plasma
membrane contains an ATP-dependent GSH transport system that is
triggered by the initiation of apoptosis. When GSH is actively pumped
out of the cell, the cytosol is shifted
from a reducing to an oxidizing
environment1.
The
ApoAlert
Glutathione Detection Kit includes a
monochlorobimane (MCB) dye that
fluoresces blue when bound to glutathione.
The
decrease
in
gluthathione levels between nonapoptotic and apoptotic cell extracts
is easily detected using a fluorometer
or 96-well fluorometric plate reader.

UV Irradiation

Resuspend in lysis buffer


& incubate

3,000
2,500
2,000

Centrifuge & collect supernatant

1,500
1,000

Control
+ Staurosporine [1 M]

500
0
0

Time after induction (hr)

MCB

Incubate cell lysate with MCB

250 ng/ml Human Fas mAb


4,000

Fluorescence units

Study redox changes in early


stages of apoptosis
Simple, quantitative in vitro assay
Easily adaptable to a 96-well format

3,500
3,000
2,500
2,000
1,500

MCB

1,000

GSH

Control
+ Fas mAb [500 ng/ml]

500

Measure blue fluorescence

0
0

Time after induction (hr)

Figure 1. Intracellular GSH levels drop in Jurkat cells


after induction of apoptosis with different agents. For
each experiment, apoptosis was induced using the indicated method in Jurkat cells. Uninduced and apoptotic
cells were incubated at 37C, samples were taken at different timepoints, and intracellular glutathione levels were
measured according to the protocol.

Figure 2. The ApoAlert Glutathione Detection assay.

Description

Format

Apps.

Size

Cat. No.

ApoAlert Glutathione Detection Kit

Kit

FA, SF

25 assays
100 assays

K2014-1
K2014-2

Cell Lysis Buffer


Cell Wash Buffer
Monochlorobimane (MCB)

References
1.van den Dobbelsteen DJ, Nobel CS, Schlegel J, Cotgreave IA, Orrenius S, Slater AF. 1996. Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody. J Biol Chem. 271(26):15420-7.

46

www.bdbiosciences.com

BD Clontech

4.5. ApoAlert Cell Fractionation Kit

tosis (1, 2). This soluble protein is


localized in the space between the
inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from
the mitochondria into the cytosol
where it initiates the caspase cascade
by binding to Apaf-1. The
cytochrome c/Apaf-1 complex activates caspase-9, which then activates
caspase-3 and other downstream
caspases. The ApoAlert Cell
Fractionation Kit allows you to separate a mitochondrion enriched fraction from the cytosol with just two
standard centrifugation steps. The
kit also includes two antibodies for
visualizing the fractions. The COX4
Antibody detects cytochrome c oxidase subunit IV (COX4), a protein

Determine the apoptotic pathway


in your model system
No ultracentrifugation required
Includes antibodies to easily distinguish fractions
The ApoAlert Cell Fractionation
Kit provides an effective way to isolate a highly enriched mitochondrial
fraction from the cytoplasm of apoptotic and non-apoptotic cells. With
this kit, you can easily determine if
cytochrome c has been released from
the mitochondria and is present in
the cytosolic fractionan indicator
of mitochondrial involvement in
apoptosis. The procedure is fast and
simple, with no ultracentrifugation
required. Cytochrome c has been
shown to play a major role in apop-

A
Fraction:
Mito
kDa

Cyt

Cyt

Cyt

Mito

kDa

kDa

43

43

43

Mito

Figure 1. Western blot detection of cytochrome c


oxidase subunit IV and cytochrome c. Apoptosis was
induced with staurosporine in NIH/3T3 cells. Six hours
after induction, the mitochondrial and cytosolic fractions
were isolated using the ApoAlert Cell Fractionation Kit.
Panel A. Mitochondrial and cytosolic fractions from
induced cells; probed with COX4 Antibody. Panel B.
Cytosolic fractions from induced and uninduced cells;
probed with Cytochrome c Antibody. Panel C.
Mitochondrial fractions from induced and uninduced
cells; probed with Cytochrome c Antibody.

29
29

29
18
18

14

cytochrome c

COX4

18
14

14
Induction:

that is localized to the inner mitochondrial membrane and remains in


the mitochondria during apoptosis.
This antibody allows you to determine the mitochondrion-enriched
fraction (Figure 1, Panel A). The second antibody is the Cytochrome c
Antibody. With this antibody you
can determine which fraction contains cytochrome c by probing
Western blots of cytosolic and mitochondrial fractions (Figure 1B &
1C). A positive result for the cytosolic fraction indicates that cytochrome
c was released from the mitochondria. The BD Clontech Cell
Fractionation Kit provides optimized
reagents and antibodies for 25 or
100 assays.

Description

Format

Apps.

Size

Cat. No.

ApoAlert Glutathione Detection Kit

Kit

FA, SF

25 assays
100 assays

K2014-1
K2014-2

Cell Fractionation Buffer


Cell Wash Buffer
Protease Inhibitor
DTT
Cytochrome c Antibody
COX4 Antibody

References
1. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. 1996 Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 12;86(1):147-57.
2. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic
protease cascade. Cell. 91(4):479-89.

BD Clontech

www.bdbiosciences.com

47

4.6. ApoAlert Mitochondrial Membrane Sensor Kit

Detect changes in mitochondrial


membrane potential
Suitable for flow cytometry or
fluorescence microscopy
Easy to use and interpret
The BD Clontech ApoAlert
Mitochondrial Membrane Sensor Kit
provides a fluorescence-based assay
for detecting apoptotic cells during
the early stages of apoptosis. The
simple procedure takes just 20 minutes to perform. Research has shown
that a cells mitochondrial transmembrane potential changes following

induction of apoptosis, resulting in


altered membrane permeability1.
MitoSensor, the key component of
this kit, is sensitive to these changes
in permeability. MitoSensor is a
cationic dye that fluoresces differently in apoptotic and nonapoptotic
cells. Normally, MitoSensor is taken
up in the mitochondria, where it
forms aggregates that exhibit intense
red fluorescence. In apoptotic cells,
MitoSensor cannot accumulate in
the mitochondria because of altered
mitochondrial membrane permeability. As a result, the dye remains in

monomeric form in the cytoplasm,


where it fluoresces green. The fluorescence signals are easily distinguished by fluorescence microscopy
or flow cytometry. The ApoAlert
Mitochondrial Membrane Sensor Kit
is also useful for studies involving
changes in mitochondrial membrane
potential that result from other cellular processes. It provides a simple,
quick method for obtaining quantitative data concerning mitochondrial membrane potential changes.

700

Apoptotic
Nonapoptotic

Cells

560
420
280
140
0
100

101

102

103

104

Green fluorescence

Figure 1. MitoSensor staining of HeLa cells. Panel A. HeLa cells were rinsed with serum-free media, stained with
MitoSensor at 37C for 20 min, rinsed once with incubation buffer, and analyzed by fluorescence microscopy using a bandpass filter. Non-apoptotic cells exhibit an intense red fluorescence. Panel B. Cells were treated with 1 M staurosporine prior
to staining with MitoSensor. Apoptotic cells exhibit a bright green fluorescence.

Figure 2. MitoSensor staining of Jurkat cells. Cells


were exposed to UV light for 5 min, incubated at 37C for
4 hr, and then stained and rinsed in suspension. Apoptotic
cells were detected using flow cytometry.

Description

Format

Apps.

Size

Cat. No.

ApoAlert Mitochondrial Membrane Sensor Kit


MitoSensor Reagent
Incubation Buffer

Kit

FA, FC

100 assays

K2017-1

References
1.Green DR, Reed JC.1998. Mitochondria and apoptosis. Science.281(5381):1309-12. Review.
The ApoAlert Mitochondrial Membrane Sensor Kit was cited in the following articles:
Biswal SS, Datta K, Shaw SD, Feng X, Robertson JD, Kehrer JP. 2000. Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis
in lipoxygenase-deficient FL5.12 cells. Toxicol Sci.53(1):77-83.
Donnelly ET, O'Connell M, McClure N, Lewis SE. 2000. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum
Reprod.15(7):1552-61.
Zhu J, Chen X. 2000 MCG10, a novel p53 target gene that encodes a KH domain RNA-binding protein, is capable of inducing apoptosis and cell cycle arrest in G(2)-M. Mol Cell Biol.
20(15):5602-18.

48

www.bdbiosciences.com

BD Clontech

4.7. Annexin V Reagents and Kits

Apoptosis is characterized by a variety of morphological features.


Changes in the plasma membrane
are one of the earliest of these features. In apoptotic cells, the
membrane phospholipid phosphatidylserine (PS) is translocated
from the inner to the outer leaflet of
the plasma membrane, thereby
exposing PS to the external cellular
environment. Annexin V is a 35-36
kD Ca2+-dependent phospholipidbinding protein that has a high affinity for PS, and binds to cells with
exposed PS. Annexin V may be conjugated to fluorochromes such as
FITC and PE, or to Biotin or fused to
EGFP (enhanced green fluorescent
protein). These formats retain their
high affinity for PS and thus serve as
sensitive probes for flow cytometric
analysis of cells that are undergoing
apoptosis. Because externalization of
PS occurs in the earlier stages of
apoptosis, Annexin V staining can

identify apoptosis at an earlier stage


than assays based on nuclear changes
such as DNA fragmentation.
Annexin V staining precedes the loss
of membrane integrity which accompanies the latest stages of cell death
resulting from either apoptotic or
necrotic processes. Therefore, staining with Annexin V in conjunction
with vital dyes such as Propidium
iodide (PI) or 7-amino-actinomycin
D (7-AAD) allows the investigator to
identify early apoptotic cells
(Annexin V positive, PI negative).
The assay does not distinguish
between cells that have already
undergone an apoptotic death and
those that have died as a result of a
necrotic pathway, because in either
case the dead cells will stain with
both Annexin V (positive) and PI
(positive).

101

102

103

103 104
102
101

Propidium Iodide

104

100

Annexin V-FITC

M2

100

101

102

101

102

103

104

Annexin V-FITC

M1

Treated with
Human Fas
mAb

Cell Frequency

100

M1

100

103 104
101

102

B Treated with
Human Fas
mAb

100

Cell Frequency

Propidium Iodide

A
A Untreated

BD Biosciences offers Annexin V


reagents individually or conveniently
packaged in our Annexin Apoptosis
Detection Kits:
The reagents of the Annexin V-FITC
Detection Kit I and the Annexin VFITC Detection Kit II are flow
cytometry based for quantitative
analysis of apoptotic cells.

Treated with
Human Fas
mAb
+ Purified Annexin V

M1

M1
M2

M2

M2

103

104

100

101

102

103

104

Annexin V-FITC

Flow cytometric analysis of apoptotic cells using Annexin V-FITC. HBP-ALL leukemia
cells were left untreated (A) or treated for 2 hr (B) with anti-human Fas antibody, clone DX2
(Cat. No. 555670) and Protein G*. Cells were incubated with Annexin V-FITC (Cat. No.
65874X or 556419) in a buffer containing Propidium iodide (PI), (Cat. No. 556463) and analyzed by flow cytometry. Untreated cells were primarily Annexin V-FITC and PI negative (inset
A), indicating that they were viable and not undergoing apoptosis. After a 2 hr treatment
with DX2, the majority of cells were undergoing apoptosis (Annexin V-FITC positive and PI
negative; inset B). The M1 and M2 gates demarcate Annexin V-FITC negative and positive
populations, respectively.

100

101

102

103

104

100

101

102

103

104

Annexin V-FITC

Flow cytometric analysis of Annexin V-FITC staining and blocking with recombinant
Annexin V. Jurkat T cells were induced to undergo apoptosis by treatment with anti-human
Fas antibody, clone DX2 (Cat. No. 555670) and Protein G* for 3 hr. Cells were then incubated with Annexin V-FITC (Cat. No. 556420 or 556419) alone (A) or with Annexin V-FITC
in the presence of recombinant Annexin V (Cat. No. 556416) (B) to block Annexin V-FITC
binding sites, thus demonstrating the specificity of Annexin V-FITC staining. *The addition of
Protein G enhances the ability of DX2 to induce apoptosis, presumably by cross-linking Fas
receptors.

www.bdbiosciences.com

49

4.7. Annexin V Reagents and Kits (continued)

Cell Frequency

Annexin V-PE
M2
M1

M2
M1

M2
M1

M2
M1

M2
M1

0h

M2
M1

M2
M1

M2
M1

1h

2h

Control

M2
M1

M2
M1

3h

4h

Camptothecin-treated

Comparison of Annexin-V and Active Caspase-3 Assays: The Annexin V assay is perhaps the most widely used assay today,
facilitated by the observation that PS translocation appears to be a universal apoptotic phenomenon. It has been detected in
mammalian, insect, and plant cells under the action of most, if not all, triggers of apoptosis. Although the Annexin V and
Active Caspase-3 assays are based on different biological phenomena, they are both used to identify cells that are in the early
stages of apoptosis .

200

Counts

160
120
uninduced

induced with
anti-Fas

80
40
0
100

101

102

103

EGFP binding
Figure 1. ApoAlertTM Annexin V-EGFP generates a bright green fluorescent signal. Jurkat cells were incubated with 200
ng/ml human fas monoclonal antibody (clone CH-11) for 8 hr to induce apoptosis. Cells were cen-trifuged, resuspended in
1X Binding Buffer, and incubated with 1 ml of Annexin V-EGFP for 5 min in the dark. Panel A. Detection of Annexin V-EGFP
by fluorescence microscopy. Positive cells exhibit green fluorescence around the plasma membrane. Panel B. Detection of
Annexin V-EGFP by flow cytometry.

Notice to Purchaser
The Annexin V-EGFP reagent is covered by U.S. Patents #5,491,084
and 5,066,787, and is the subject of pending patent applications. All
purchasers are granted an automatic license with the purchase of the
Annexin V-EGFP reagent to use it for internal research purposes.
However, this license excludes specifically any right to modify the
reagent for resale, to use the reagent for the manufacture of commercial products, or to use the reagent in humans or for diagnostic
purposes. Certain Living Colors products, and other products containing "enhanced" GFP variants, are covered by the following license
terms. Use of CLONTECH's Living Colors products containing DNA
sequences coding for mutant Aequorea victoria green fluorescent protein (GFP) variants or proteins thereof requires a license from Aurora
Biosciences Corporation under U.S. Patent Nos. 5,625,048, 5,777,079,
6,054,32 and 5,804,387 and other pending U.S. and foreign patent
applications. In addition, certain CLONTECH products are made under
U.S. Patent No. 5,804,387 licensed from Stanford University. Not-For-

50

www.bdbiosciences.com

Active
Caspase-3-FITC

Profit research institutes or entities are granted an automatic license


with the purchase of this product for use in non-commercial internal
research purposes, the terms of which are disclosed in detail in the
license that accompanies the shipment of this product. Such license
specifically excludes the right to sell or otherwise transfer this product
or its components to third par-ties. For-Profit research institutes or entities that wish to use this product in non-commercial or commercial
applications are required to obtain a license from Aurora Biosciences
Corporation. For license information contact: Court Turner at 858-4048416 or Fax 858-404-6743 or www.aurorabio.com. Please contact
CLONTECH directly for any other assistance, including purchasing and
technical support. All companies and institutions purchasing Living
Colors products will be included in a quarterly report to Aurora
Biosciences Corporation, as required by the CLONTECH/Aurora license
agreement.

The ApoAlert Annexin V-FITC


Apoptosis Kit and the ApoAlert
Annexin V-EGFP Apoptosis Kit
assay is a one-step procedure that
takes just 10 minutes to perform and
can be used to detect apoptosis by
fluorescence microscopy or flow
cytometry. The assay is nonenzymatic, does not require fixation, and
is suitable for both adherent and suspension cells.
ApoAlert Annexin V-EGFP is the
brightest green fluorescent reagent
available for annexin V-based detection of apoptosis. Enhanced green
fluorescent protein (EGFP) is
extraordinarily bright and resistant
to photobleaching, making it the
best reagent for experiments that
require high sensitivity, such as fluorescence microscopy (Figure 1A).
Annexin V-EGFP can also be used to
detect apoptotic cells by flow cytometry (Figure 1B). Because Annexin VEGFP is a fusion protein, it has a 1:1
ratio of EGFP to PS and so the
results obtained from the assay are
quantitative. ApoAlert Annexin VEGFP is available separately or in a
complete kit. The kit can be formatted for 96-well assays for highthroughput analysis.

Product Listing

Description

Format/Conc.

Applications Size

Cat. No.

New Cat. No.

Recombinant Annexin V (For Blocking)


Annexin V-Biotin
Annexin V-Biotin
Annexin V-FITC
Annexin V-FITC
ApoAlert Annexin V-FITC
Annexin V-PE
Annexin V-PE
Annexin V-APC
Annexin V-APC
Annexin V-Cy5
Annexin V-Cy5
Annexin V-Cy5.5
Annexin V-Cy5.5
ApoAlert Annexin V-EGFP
Annexin V-FITC Detection Kit I
Annexin V-FITC Detection Kit II
ApoAlert Annexin V-FITC Apoptosis Kit
ApoAlert Annexin V-FITC Apoptosis Kit
ApoAlert Annexin V-EGFP Apoptosis Kit
ApoAlert Annexin V-EGFP Apoptosis Kit
Annexin V Binding Buffer
ApoAlert Annexin V 10x Binding Buffer
Propidium Iodide Solution
ViaProbe (7-AAD) Staining Solution

Unlabeled
Biotin
Biotin
FITC
FITC
FITC
PE
PE
APC
APC
Cy5
Cy5
Cy5.5
Cy5.5
EGFP tagged
Kit
Kit
Kit
Kit
Kit
Kit
Buffer
Buffer
Buffer
Buffer

65871A
65872H
65872X
65874H
65874X

ViaProbe (7-AAD) Staining Solution


Annexin V-FITC Fluorescence Microscopy

Buffer
Kit

FC
FC
FC
FC
FC
FC, FM
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC, FM
FC, FM
FC
FC
FC
FC, FM
FC
FC
FC
FC
FM

556416
556417
556418
556419
556420
8133-1
556421
556422
550475
550474
559934
559933
559936
559935
8137-1
556547
556570
K2025-1
K2025-2
K2019-1
K2019-2
556454
8134-1
556463
555816
555815
559925
550911

0.1 mg
200 tests
100 tests
200 tests
100 tests
500 assays
200 tests
100 tests
200 tests
100 tests
200 tests
100 tests
200 tests
100 tests
500 assays
100 tests
100 tests
50 assays
200 assays
50 assays
200 assays
50 ml
100 ml
2.0 ml
100 tests
500 tests
2ml

65875H
65875X
65879H
65879X
6587MH
6587MX
6587NH
6587NX
6693KK
6710KK

66121E
66211E
34321X
34321J
68981E
8065KK

Annexin V-FITC Detection Kit I, ApoAlert Annexin V-FITC Apoptosis Kit and ApoAlert Annexin V-EGFP Apoptosis Kit
Kit Components
Annexin V-FITC or Annexin V-EGFP
Binding Buffer
Propidium Iodide
User Manual

Annexin V-FITC Detection Kit II


Kit Components
100g Recombinant Annexin V
Annexin V-FITC
Binding Buffer
Propidium Iodide
User Manual

The ApoAlert Annexin V-FITC Apoptosis Kit was cited in the following articles:
Takao N, Mori R, Kato H, Shinohara A, Yamamoto K. 2000. c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated functions in chromosomal maintenance. J Biol Chem.
275(2):725-8.
Maeshima Y, Colorado PC, Torre A, Holthaus KA, Grunkemeyer JA, Ericksen MB, Hopfer H, Xiao Y, Stillman IE, Kalluri R. 2000. Distinct antitumor properties of a type IV collagen domain
derived from basement membrane. J Biol Chem. 275(28):21340-8.
Persad S, Attwell S, Gray V, Delcommenne M, Troussard A, Sanghera J, Dedhar S. 2000. Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell
cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci U S A.97(7):3207-12.
The ApoAlert Annexin V-EGFP Apoptosis Kit was cited in the following articles:
Shaoqiong Chen, Denis C. Guttridge, Zongbing You, Zhaochen Zhang, Andrew Fribley, Marty W. Mayo, Jan Kitajewski, and Cun-Yu Wang. 2001. Wnt-1 Signaling Inhibits Apoptosis by
Activating b-Catenin/T Cell Factormediated Transcription. J. Cell Biol. 152(1):8796
Yi Ting Zhou, Unice J.K. Soh, Xun Shang, Graeme R. Guy and Boon Chuan Low. 2002. The BNIP-2 and Cdc42GAP Homology / Sec-14p-like domain of BNIP-Sa a is a novel apoptosis-inducing sequence. J. Biol. Chem. In press

BD Pharmingen
BD Clontech

www.bdbiosciences.com

51

4.8. ApoAlert Nitric Oxide/Annexin V Dual Sensor Kit

A simple fluorimetric
method for measuring
nitric oxide in apoptotic
cells

binding NO 3, 4. The NO-sensing dye


readily diffuses into living cells
where it serves as a direct reporter of
nitric oxide synthase (NOS) activity.

Measure nitric oxide synthesis in


real time
Study the link between nitric
oxide production and apoptosis
Ideally suited for FACS analysis

Simultaneous detection of
apoptotic events

The BD Clontech ApoAlert Nitric


Oxide/Annexin V Dual Sensor Kit is
a new combination kit that provides
a simple, fluorescence-based method
for correlating nitric oxide (NO) synthesis with one of the key events in
apoptosisthe exposure of phosphatidylserine (PS) on the plasma
membrane.

Nitric oxide synthesis and


apoptosis
The Nitric Oxide/Annexin V Dual
Sensor Kit addresses a relatively new
question in apoptosis research: What
role does nitric oxide play in programmed cell death?1, 2. Whereas low
levels of NO synthesis are beneficialeven essentialfor cellular
activities such as memory formation,
tumor suppression, immunity, and
vasodilation, excessive NO destroys
cells by disrupting energy metabolism and damaging DNA. Studies
have shown that apoptotic cells produce NO at a much faster rate than
healthy cells, a finding that has
prompted many researchers to ask
whether a cells fate can be governed
by regulating NO synthesis1. That
question is the basis for the BD
Clontech Nitric Oxide/Annexin V
Dual Sensor Kit, which enables you
to monitor NO synthesis before, during, and after the onset of apoptosis.
Our method makes use of a recently
developed, membrane-permeable
dye that fluoresces bright green upon

52

www.bdbiosciences.com

To detect the onset of apoptosis, the


kit also contains annexin V-phycoerythrin (PE), a protein-fluorophore
conjugate with a strong, specific
affinity for phosphatidylserine (PS).
Annexin V binds PS when it flips
from the inner to the outer leaflet of
the plasma membrane. Cells reaching this critical apoptotic stage fluoresce bright red because of the
phycoerythrin label joined to
annexin V. Because of their distinct
emission spectra, the NO-sensing
dye and annexin V-phycoerythrin
can be used together to follow NO
production and PS externalization in
the same cell. With flow cytometry,
the green and red signals are clearly
distinguished on separate channels
so you can follow both processes
simultaneously (Figure 2).

Simple fluorescence-based
assay
It is easy to prepare cells for the
NO/Annexin V assay (Figure 1). The
assay is nonenzymatic, requires no
fixatives, and is suitable for adherent or non-adherent cells. First, incubate cells with the NO-sensing dye
for 30 minutesno washing or rinsing steps are requiredthen induce
apoptosis using your method of
choice. You can assay nitric oxide
and PS externalization separately or
together. In either case, simply collect
cells at preselected intervals, resuspend in buffer (with or without
annexin V-phycoerythrin) and analyze by flow cytometry.

We have used the kit to track NO


production and PS externalization in
Jurkat, 3T3, and HeLa cells treated
with UV lighta powerful apoptotic stimulus. In each case, the assay
clearly showed how cells respond to
the stimulus by boosting their rates
of NO production (Figure 3). Our
kit is ideally suited to the cell-by cell
analysis possible with flow cytometry. By using the annexin V and NO
Sensors together, you can estimate
cell heterogeneity (Figure 2) and separate selected subpopulations for

further analysis with a third apoptotic marker . The NO Sensor Dye is


not only easy to use, its also
extremely specific and highly sensitive. When the fluorophore binds
NO, its quantum yield increases
nearly 100-fold. As a result, you can
detect as little as 5 nM NO5. This
means you can monitor a full range
of NOS activity inducible and constitutivein all cell types, including
endothelial cells, nerve cells, and
macrophages1.

Add NO-Sensor Dye to Cell Culture


Incubate for 30 min at 37C
Induce apoptosis

Incubate for desired time


Centrifuge & collect pellet

Annexin V
PE

Resuspend in
Binding Buffer

Resuspend in Binding Buffer,


PBS, or FACS Sheath Fluid
(FACSFlow)

Incubate for
15 min at RT

FACS

FACS

(FL-1 & FL-2)

(FL-1)

Figure 1. Measuring nitric oxide in cultured cells. The NO Sensor Dye (Excitation max 495 nm; Emission max 515 nm) fluoresces approximately 100 times brighter after binding nitric oxide5. Although the dye readily enters cells, it can not exitcellular esterases lock the dye inside by removing its hydrophobic tail. Annexin V-phycoerythrin (PE) binds phosphatidylserine
when it flips to the outer leaflet of the plasma membrane, a hallmark of apoptotic cells.

www.bdbiosciences.com

53

4.8. ApoAlert Nitric Oxide/Annexin V Dual Sensor Kit


(continued)

6 hr

UV

+ UV

UV

+ UV

6 hr

24 hr

B
B

6 hr

24 hr
24 hr
Figure 3. UV light causes a surge in nitric oxide synthesis. Cell cultures were incubated with the NO Sensor Dye (5 M)
for 30 min, exposed to UV light for 5 min, and then incubated at 37C for the indicated times. Green fluorescence was
measured by flow cytometry. Panel A. HeLa cells. Panel B. Jurkat cells. 3T3 cells were also tested with similar results (not
shown).

Figure 2. Using flow cytometry to follow nitric


oxide production and phosphatidylserine externalization in HeLa cells. Cells were incubated with
the NO Sensor Dye (5 M) for 30 min, exposed to
UV light for 5 min, and then incubated at 37C for
the indicated times. Cells were then resuspended
in Annexin V Binding Buffer containing annexin
V-PE and incubated at room temperature for 15
min before scanning with flow cytometry. Green
and red fluorescent emissions were read using the
FL-1 and FL-2 channels, respectively. The analysis
shows that nitric oxide synthesis and phosphatidylserine externalization do not occur at the
same time in the same cells (Panel A & Panel B).
With FACS, these subpopulations can be separated and further characterized with a third apoptotic marker.

Description

Assays

Cat. No.

ApoAlert Nitric Oxide/ Annexin V-PE Dual Sensor Kit

25
100

K2013-1
K2013-2

Components
Nitric Oxide Sensor Dye
Annexin V-Phycoerythrin (PE)
Annexin V Binding Buffer

References
1. Ushmorov A, Ratter F, Lehmann V, Droge W, Schirrmacher V, Umansky V. 1999. Nitric-oxide-induced apoptosis in human
leukemic lines requires mitochondrial lipid degradation and cytochrome C release. Blood. 93(7):2342-52
2. Murphy MP. 1999. Nitric oxide and cell death. Biochim Biophys Acta. 1411(2-3):401-14. Review.
3. Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, Maeda D, Imai Y, Irimura T, Nagano T. 1998. Direct evidence of
nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS
Lett. 427(2):263-6.
4. Kojima H, Sakurai K, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. 1998. Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore. Chem Pharm Bull (Tokyo).46(2):373-5.
5. Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. 1998. Detection and imaging of
nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem. 70(13):2446-53.

54

www.bdbiosciences.com

BD Clontech

APO-BRDU Assay

4.9. BD Biosciences TUNEL Assays


4.9.1. APO-DIRECT and APO-BRDU Kits
Relative Cell Number

The APO-BRDU assay usues bromolated


deoxyribonucleotide
triphosphates (Br-dUTP) to label the
3-hydroxyl (OH) termini of doubleand single-stranded DNA5. It has
been shown that Br-dUTP is more
readily incorporated into the genome
of apoptotic cells than are the
deoxyribonucleotide triphosphates
complexed to larger molecules. After
incorporation, these sites are identified by flow cytometric means by
staining the cells with a FITC-labeled
anti-BrdU mAb.

M1
M2

100

101

10 2

B
Relative Cell Number

APO-BRDU*

The Apo-DIRECT assay is a single-step method for labeling DNA


breaks with FITC-dUTP followed by
flow cytometry analysis6.

M2

10

10

10

55

10

10

Treated with
Human Fas
mAb (12 hr)
M1 = 34%
M2 = 66%

M1
M2

M1

* APO-BRDU and APO-DIRECT are registred trademarks of Phoenix Flow Systems.

104

M1

10

References
1. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu and S. Nagata. 1998. A caspase-activated DNase that
degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 394:43-50.
2. Sakahira, H., M. Enari and S. Nagata. 1998. Cleavage of CAD inhibitor in CAD activation and DNA degradation during
apoptosis. Nature. 391:96-99
3. Walker, R.P., L. Kokileva, J. LeBlanc and M. Sikorska. 1993. Detection of the initial stages of DNA fragmentation in apoptosis. Biotechniques 15:1032-1036.
4. Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami and T. Traganos, T. 1997. Cytometry in cell necrobiology:
Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1-20
5. Li, X., F. Traganos, M.R. Melamed, and Z. Darzynkiewicz. 1995. Single step procedure for labeling DNA strand breaks
with fluorescein- or BODIPY-conjugated deoxynucleotides. Detection of apoptosis and bromodeoxyuridine incorporation.
Cytometry 20:172-180.
6. Li, X. and A. Darzynkiewicz. 1995. Labelling DNA strand breaks with Brd-UTP. Detection of apoptosis and cell proliferation Cell Prolif. 28:572-579

10 3

Treated with
Human Fas
mAb (2 hr)
M1 = 63%
M2 = 37%

APO-DIRECT*

www.bdbiosciences.com

Untreated
M1 = 99%
M2 = 1%

Relative Cell Number

One of the later steps in apoptosis is


DNA fragmentation, a process
which begins with the activation of
endonucleases which become activated during the apoptotic program1, 2.
These nucleases degrade the higher
order chromatin structure into fragments of 50 to 300 kb and subsequently into smaller DNA pieces of
about 200 bp length. A method
which is often used to detect fragmented DNA utilizes a reaction catalyzed by exogenous TdT, referred to
as end-labeling or TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick-end-labeling)4
staining.
The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes
a template independent addition of
labeled deoxynucleotides.

HPB-ALL Human Leukemia Cells

10

10
BrdU-FITC

10

10

APO-BRDU Kit, Cat. No. 6576KK. HBP-ALL


human leukemia cells were left untreated (A) or
treated with anti-human Fas mAb, clone DX2
(Cat. No. 33450D), and Protein G* for 2 hr (B) or
12 hr (C). Cells were fixed and incubated with BrdUTP** in the presence of TdT enzyme** in
order to incorporate Br-dUTP into exposed 3-OH
DNA ends. Br-dUTP was detected with a fluorescein labeled anti-BrdU mAb**. Nonapoptotic
cells (M1 gates) do not incorporate significant
amounts of Br-dUTP due to lack of exposed 3OH DNA ends, and consequently have relatively
little fluorescence compared to apoptotic cells
which have an abundance of 3-OH ends (M2
gates). DX2 induced Fas-mediated apoptosis is
shown by an increase in the number of cells
staining with anti-BrdU-FITC mAb (M2 gates)
after 2 and 12 hr. The M1 and M2 gates demarcate non-apoptotic and apoptotic populations,
respectively.
*The addition of Protein G enhances the ability
of DX2 to induce apoptosis, presumably by crosslinking Fas receptors.
**Components of the APO-BRDU Kit.

4.9.2. ApoAlert DNA Fragmentation Assay Kit


A

The BD Biosciences Clontech


ApoAlert DNA Fragmentation
Assay Kit incorporates fluoresceindUTP at the free 31- hydroxyl ends of
the fragmented DNA1. The assay can
be used with tissue sections as well as
cultured cells, and results can be
visualized directly by fluorescence
microscopy or quantified by flow
cytometry. (Figure 2).
Detect nuclear DNA fragmentation in situ
Use with adherent cells, suspension cells, or fixed tissue sections
Analyze results by fluorescence
microscopy or flow cytometry

50
Counts

50
Counts

Figure 1. DNA fragmentation in tissue sections.


Mouse embryo (day E14) tissue was fresh-frozen,
cut into sections, and mounted on slides by standard methods. The ApoAlert DNA Fragmentation
Assay was performed according to the User
Manual. Sections were photographed using a fluorescence microscope with either a propidium
iodide (PI) filter alone (Panel A), a FITC filter alone
(Panel B), or a dual-pass FITC/PI filter set (Panel C).
Apoptotic cells appear green with the FITC filter
alone. Apoptotic cells appear yellow and nonapop-totic cells appear red under the dual-pass
FITC/PI filter set.

40
30
20

40
30
20
10

10

100

101

103
102
FITC Fluorescence Intensity

104

100

101

102
103
FITC Fluorescence Intensity

104

Figure 2. FACS analysis of DNA fragmentation in apoptotic cells in suspension. Jurkat cells were grown in RPMI 1640
medium + 10% FBS. Apoptosis was induced by treatment with 200 ng/ml anti-Fas mAb for 15.5 hr. The ApoAlert DNA fragmentation assay was performed according to the User Manual. Cells were analyzed by flow cytometry. Panel A. Uninduced
cells. Panel B. Induced cells.

56

www.bdbiosciences.com

Product Listing
Description

Apps.

Format

Size

Cat. No.

New Cat. No.

APO-BRDU KIT
Fluorescein Labeled Anti-BrdU mAb
PI/Rnase Staining Buffer
Reaction, Rinsing and Wash Buffer
Br-dUTP
Negative/Positive Control Cells
TdT Enzyme

FC

Kit

60 tests

6576KK

556405

APO-DIRECT KIT
PI/Rnase Staining Buffer
Reaction, Rinsing and Wash Buffer
FITC-dUTP
Negative/Positive Control Cells
TdT Enzyme

FC

Kit

50 tests

6536KK

556381

ApoAlert DNA Fragmentation Assay Kit

IF, FC

Kit

25 assays
100 assays

K2024-1
K2024-2

Equilibration Buffer
Nucleotide Mix
TdT Enzyme
SSC
Proteinase K
Plastic Coverslips

References
1. Gavrieli Y, Sherman Y, Ben-Sasson SA. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. Nov;119(3):493-501.
The ApoAlert DNA Fragmentation Assay Kit was cited in the following articles:
Christelle Forcet, Xin Ye, Laure Granger, Vronique Corset, Hwain Shin, Dale E. Bredesen, and Patrick Mehlen. 2001. The dependence receptor DCC (deleted in colorectal cancer) defines an
alternative mechanism for caspase activation. Proc. Natl. Acad. Sci. 98(6):3416-3421
Prasad Devarajan, Maryely De Leon, Farahnaz Talasazan, Alan R. Schoenfeld , Eliot J. Davidowitz , and Robert D. Burk. 2001. The von Hippel-Lindau Gene Product Inhibits Renal Cell
Apoptosis via Bcl-2-dependent Pathways. J. Biol. Chem. 276( 44):4059940605
Srigiridhar Kotamraju, Neil Hogg, Joy Joseph, Larry K. Keefer, and B. Kalyanaraman. 2001. Inhibition of Oxidized Low-density Lipoprotein-induced Apoptosis in Endothelial Cells by Nitric
Oxide J. Biol. Chem. 276(20):17316-17323

BD Pharmingen
BD Clontech

www.bdbiosciences.com

57

4.10. ApoAlert LM-PCR Ladder Assay Kit

Detect nucleosomal ladders even


in samples containing only small
fractions of apoptotic cells
Suitable for small samples, such as
biopsies
Compare relative levels of apoptosis in different samples
The ApoAlert LM-PCR Ladder
Assay Kit provides a sensitive
method for detecting nucleosomal
ladders generated during apoptosis 1, 2.

24-mer
HOHO-

Based on ligation-mediated PCR


(LM-PCR3), the kit specifically
amplifies the nucleosomal ladder,
allowing this apoptotic event to be
visualized. Moreover, the LM-PCR
assay is semiquantitative, so the relative extent of apoptosis in different
samples can be compared. The BD
Clontech ApoAlert LM-PCR Ladder
Assay Kit includes the necessary
reagents for performing 50 reactions.

5'

3'
-OH HO-P
HO5'

-OH P-OH HO-

12-mer

3'

-OH

Fill in ends

Amplify by PCR (1530 cycles)

Resolve amplified fragments


on agarose/EtBr gel

800
600
400
200
Figure 1. The ApoAlert LM-PCR assay. Apoptosis was induced in Jurkat cells by incubation with 200 ng/ml human Fas monoclonal antibody (clone CH-11) for 9 hr. Genomic
DNA was isolated from uninduced and induced Jurkat cells. The LM-PCR assay was performed according to the User Manual (15 PCR cycles). 15 l of each reaction was electrophoresed on a 1.2% agarose/EtBr gel. Lane 1: uninduced. Lane 2: induced (apoptotic).

58

-OH

Ligate adaptor to DNA fragments


Heat to release 12-mer

www.bdbiosciences.com

Product Listing

Description

Apps.

Format

Size

Cat. No.

ApoAlert LM-PCR Ladder Assay Kit


Ligation Mix
T4 DNA Ligase
LM-PCR Mix
Control Mix
Control Calf Thymus DNA
Mouse & Human En-2 Control Templates & Primer Sets

Kit

FA

50 rxns

K2021-1

Notice to Purchaser
These products are optimized for use in the Polymerase Chain Reaction
(PCR) covered by patents owned by Hoffmann-La Roche and F.
Hoffmann-La Roche Ltd. Under these patents no license to use the PCR
process is conveyed expressly or by implication to the purchaser by the
purchase of these products. A license to use the PCR process for certain research and development activities accompanies the purchase of
certain reagents from licensed suppliers, such as CLONTECH
Laboratories, Inc., when used in conjunction with an authorized thermal cycler, or is available from Perkin-Elmer Corporation. Further information on purchasing licenses to practice the PCR process may be
obtained by contacting the Director of Licensing at the Perkin-Elmer
Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404 or Roche
Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501.

References
1. Wyllie AH. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature.284(5756):555-6.
2. Arends MJ, Morris RG, Wyllie AH. 1990. Apoptosis. The role of the endonuclease. Am J Pathol. 136(3):593-608.
3. Staley, K., Blaschke, A. J., and Chun, J. 1997. Apoptotic DNA fragmentation is detected by a semiquantitative ligation-mediated PCR of blunt DNA ends. Cell Death Diff. 4:66-75
The ApoAlert LM-PCR Ladder Assay Kit was cited in the following articles:
Yasunori Kasahara, Rubin M. Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras, Steven Abman, Peter K. Hirth, Johannes Waltenberger, and Norbert F. Voelkel. 2000. Inhibition of VEGF
receptors causes lung cell apoptosis and emphysema. J. Clin. Invest. 106(11):13111319
Eric Tse and Terence H. Rabbitts. 2000. Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy. Proc. Natl. Acad. Sci. 97(22):12266-12271

BD Clontech

www.bdbiosciences.com

59

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