Introduction

The proteome is exponentially more complex than the genome because diversity can be
introduced at multiple levels. These include amino acid sequences, expression levels,
post-translational modifications (e.g., glycosylation) and alternative splicing.
Protein interactions are critical to the majority of cellular processes, including replication,
transcription, translation and signal transduction. The characterization of these interactions
aids researchers in determining the function of proteins and the genes that encode them.
The methods utilized in this characterization process differ depending on the nature of the
experimental design. Techniques such as the yeast two-hybrid system are used to screen
large numbers of potential proteins that may interact with the protein of interest. Other
techniques such as pull-down assays can be used for both the discovery and characterization
of individual protein pairs.
This guide provides an overview of common techniques used for protein interaction analysis
and will assist you in determining which individual technique or combination of techniques is
best suited for a particular situation/experiment.
The methods covered include:
1. Yeast Two-hybrid System
2. Mammalian Two-hybrid System
3. Pull-down Assays
4. Co-immunoprecipitation
5. Co-localization/FRET/BRET
6. Chromatin Immunoprecipitation (ChIP)
7. Electrophoretic Mobility Shift Assay (EMSA)

Table 1. Methods and comparison selection guide.

Experimental
Primary type Experimental Goal: Cell-based Cell-free Time
of interaction Goal: Confirmation or format based format (does not include
Method detected Discovery Characterization available available vector construction)

Yeast Two-hybrid
System Protein:protein yes no yes no 8–12 days

Mammalian Two- Protein:protein no yes yes no 3–4 days

hybrid System

1–4 days
Pull-down Assays Protein:protein no yes yes yes
(depending on format)

Co- 1–4 days

Protein:protein no yes yes yes
immunoprecipitation (depending on format)

Co-localization/ Protein:protein no yes yes no 3–4 days

FRET/BRET

Chromatin 2–5 days

Protein:DNA yes yes yes no
immunoprecipitation (depending on format)

Protein:nucleic 1–4 days

EMSA yes yes yes yes
acid (depending on format)
 TABLE OF CONTENTS

Chapter Title Page

1 Yeast Two-hybrid System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
2 Mammalian Two-hybrid System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
3 Pull-down Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
4 Co-immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
5 Co-localization/FRET/BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
6 Chromatin Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
7 Electrophoretic Mobility Shift Assay (EMSA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
8 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

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PROMEGA PROTEIN INTERACTION GUIDE

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ii
Chapter One: Yeast Two-hybrid System

Contents Page

Yeast two-hybrid system overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Yeast two-hybrid system formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
1
CHAPTER

YEAST
When to use the yeast two-hybrid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 TWO-HYBRID
SYSTEM
Primary reagent requirements for the yeast two-hybrid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Yeast two-hybrid system overview

The yeast two-hybrid system is based on the fact that eukaryotic transcriptional activators
consist of two individual domains, the DNA binding domain (DBD) and the activation domain
(AD). The DBD recognizes a specific DNA sequence. The AD coordinates the assembly of the
elements required for transcription and enables RNA polymerase II to transcribe a specific
reporter gene downstream of the DBD domain.
Using the yeast two-hybrid system the protein of interest (X) is expressed as a fusion protein to
the DBD (DBD-X; also known as the “bait” protein) and the activation domain is fused to the
second protein of interest (Y), (AD-Y; also known as the “prey” protein).
The AD-Y fusion vector is introduced into a yeast strain containing the DBD-X fusion partner by
transformation or mating. Only if proteins X and Y physically interact with one another are the
DBD and AD brought together to activate expression of the downstream reporter gene (Figure 1).

PROMEGA PROTEIN INTERACTION GUIDE 1

YEAST TWO-HYBRID SYSTEM

Bait Plasmid Prey Plasmid

DBD X
AD Y

X
DBD Y AD

X Y AD
DBD Transcription
Machinery
Reporter Gene

6639MA
Figure 1. Principle of the yeast two-hybrid system. The protein coding sequence of the bait protein is cloned into a vector containing
the DNA binding sequence (DBD-X (bait) fusion). The protein coding sequence of the prey protein is cloned into a vector that contains
sequences for transcription activation (AD-Y (prey) fusion). Both vectors must also contain the necessary elements for growth and protein
expression in yeast. The recombinant vectors are introduced into the appropriate yeast strain. Only if proteins X and Y physically interact
with one another are the DBD and AD brought together to reconstitute a functionally active factor that binds to upstream specific sequences
of the reporter gene and activates expression.

REFERENCES
Key Original Reference
1. Chien, C. et al. (1991) Proc.
Natl. Acad. Sci. 88, 9578–82.
GAL4-based yeast
two-hybrid system
1. Tzeng, S-L. et al. (2006)
J. Biol. Chem. 281, 15405–11.
2. McFie, P. et al. (2006)
J. Biol. Chem. 281, 18069–80.
3. Orioli, D. et al. (2006) Mol. Biol.
Cell. 17, 2391–2400.
4. Hattori, T. et al. (2006)
J. Biol. Chem. 281, 14417–28.
5. Okoye, M. et al. (2006)
J. Virol. 80, 929–40.

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2
Yeast two-hybrid system formats
There are several varieties of the yeast two-
hybrid system. The two most commonly used
systems differ in the nature of the DBD used to
express the bait fusion protein (GAL4 or LexA),
and the AD used to generate the prey fusion
protein (GAL4, VP16 or B42).
In order to reduce the occurrence of false
positives, typically a combination of four
reporter genes (e.g., HIS3, URA3, ADE2 and
lac Z) are stably integrated in single-copy
numbers at different loci in the appropriate
yeast genome. Induction of the H153, ADE2
or URA3 reporter genes allows monitoring of
the transcription activation by growth on
plates lacking histidine, adenine or uracil.
Induction of the downstream lac Z gene
results in a blue color yeast colony when
assayed with X-Gal (5-bromo-4-chloro-3-
indolyl-D-galactopyranoside).
PROMEGA PROTEIN INTERACTION GUIDE
When to use the yeast two-hybrid system
The yeast two-hybrid system is often the first
method used to identify protein interactions. It
provides an ideal format for screening one
individual bait protein against large prey cDNA
libraries, which can be generated by the user
1
CHAPTER
YEAST
or purchased from commercial sources. Once TWO-HYBRID
tentative partners have been identified other SYSTEM
methods are used to confirm and characterize
the pair.
Primary reagent requirements for the yeast two-
hybrid system
• Yeast expression vector containing DNA
binding domain (e.g., GAL4) and
sequence coding for the bait protein
• Yeast expression vector containing
activator domain (e.g., VP16) and
sequence coding for the prey protein
• Appropriate yeast strains
• Appropriate media for yeast growth and
detection of interactions
REFERENCES (CONTINUED)
LexA-based yeast
two-hybrid system
1. Gorska, M. et al. (2006)
J. Biol. Chem. 281, 14429–39.
2. Stanasila, L. et al. (2006)
J. Biol. Chem. 281, 4354–63.
3. Yueh, A. et al. (2006)
J. Virol. 80, 342–52.
4. Zhang, Y. et al. (2006)
J. Cell. Sci. 119, 1666–76.
5. Yang, Y. et al. (2006)
J. Biol. Chem. 281, 22352–59.
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 2
CHAPTER

MAMMALIAN
Chapter Two: Mammalian Two-hybrid System

Contents

Mammalian two-hybrid system overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Mammalian two-hybrid system formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Page

TWO-HYBRID Luciferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
SYSTEM
SEAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
β-Galactosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
When to use the mammalian two-hybrid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Primary reagent requirements for the mammalian two-hybrid system . . . . . . . . . . . . . . . . . . . . . . . . . .6

Mammalian two-hybrid system overview

The mammalian two-hybrid system is similar to the yeast two-hybrid system in that both are
based on the fact that eukaryotic transcription factors are comprised of two distinct physical
and functional domains: a DNA binding domain (DBD) and an activation domain (AD). The DBD
recognizes a specific DNA sequence, in this case a promoter/enhancer element. The AD
coordinates the assembly of the elements required for transcription allowing RNA polymerase II
to transcribe a specific reporter gene downstream of the DBD.
The mammalian two-hybrid system relies upon three plasmids that are co-transfected into
mammalian cells. Each plasmid has unique features. One plasmid contains a transcriptional
activation domain upstream of coding sequences for the prey protein (AD-Y). The second
vector contains a DBD upstream of coding sequences of the bait protein (DBD-X). The third
vector contains five DNA binding sites upstream of a minimal TATA box, which is upstream of a
specific reporter gene.
Interaction between proteins “X” and “Y” result in association of the DBD with the
transcriptional activation domain. When the complex binds to the DNA binding sites on a
specially designed reporter vector, transcriptional activation of the reporter gene occurs
(expression of the two domains individually will not lead to activation of the reporter gene)
(Figure 2).

4
Mammalian two-hybrid system formats
The mammalian two-hybrid system allows
characterization of mammalian protein:protein
interactions within a cellular environment that
mimics native conditions. Yeast and
mammalian cells differ in patterns of post-
assay, typical reporter assays in the
mammalian system can be performed within
48 hours of transfection.
The most common format of the mammalian
two-hybrid system consists of one vector
containing the DBD of the GAL4 protein,
2
CHAPTER

translational modification, such as
glycosylation, phosphorylation and acylation,
as well as in the intracellular localization of
proteins. These types of protein modifications,
as well as other unique factors or modulators
present in mammalian cells, may influence the
ability of protein domains to interact.
Another advantage of the mammalian two-
hybrid system is that the assay is less time-
consuming than the yeast two-hybrid system.
Instead of waiting 3–4 days for yeast colonies
to grow to a reasonable size for a blue-color
MAMMALIAN
another vector containing the AD of the TWO-HYBRID
herpes simplex virus VP16, and a third vector SYSTEM
containing 4-5 GAL4 binding sites upstream
of a specific reporter gene.
The primary difference between the various
systems is the reporter gene used for
detection of positive interactions. The three
most commonly used reporter genes are
luciferase, β-galactosidase and secreted
alkaline phosphatase (SEAP).

DNA Binding Protein Activator Protein DNA

Domain Partner #1 Domain Partner #2 Binding Reporter
GAL4 BD (PP#1) VP16 AD (PP#2) Sequences Gene

DBD-X AD-Y

Transfect all 3 vectors into Mammalian Cells

Expression of Reporter
Gene Indicates Positive
Protein:Protein Interaction

VP16 AD
PP#2
PP#1
GAL4 BD
6542MA

DNA Binding Reporter Gene

Sequences TO ORDER
Figure 2. Principle of the mammalian two-hybrid system. The protein coding sequence for the bait protein is cloned into a vector that Phone
contains the DNA binding sequence (DBD-X (bait) fusion). The protein coding sequence for the prey protein is cloned into a vector that 1-800-356-9526
contains the sequences for transcriptional activation (AD-Y (prey) fusion). The vectors also must contain the necessary elements for growth Fax
and protein expression in mammalian cells. The recombinant vectors are then transfected along with a third plasmid containing the appro- 1-800-356-1970
priate DNA binding sites upstream of a reporter gene. Only if proteins X and Y physically interact are the DBD and AD brought together to Online
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reconstitute a functionally active factor that binds to upstream sequences and activate expression of the reporter gene.

PROMEGA PROTEIN INTERACTION GUIDE 5

MAMMALIAN TWO-HYBRID SYSTEM
REFERENCES
Key original reference for
mammalian two-hybrid system
1. Fearon, E.R. et al. (1992) Proc.
Natl. Acad. Sci. 89, 7958–62.
Using luciferase as the reporter
gene
1. Cheng, G. et al. (2006)
J. Biol. Chem, 281, 17718–26.
2. Yamaguchi, T. (2006) J. Mol.
Endocrinol. 36, 569–79.
3. Tamura, S. et al. (2006)
J. Biol. Chem. 281, 27693–704.
4. Wessels, E. et al. (2006)
J. Biol. Chem. 281, 28232–43.
5. Kato, N. et al. (2006)
J. Immunol. 177, 147–54.
Using secreted alkaline
phosphatase as a reporter gene
1. Elhaji, Y. et al. (2006) Hum. Mol.
Genet. 15, 921–31.
2. Liu, B-F. et al. (2006) J. Biol.
Chem. 281, 2624–30.
3. Lin, S. et al. (2006) J. Biol.
Chem. 281, 16716–26.
4. Cheng, G. et al. (2006) J. Biol.
Chem. 281, 17718–26.
5. Mellamed, P. et al. (2006)
Endro. 147, 3598–605.
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Luciferase When to use the mammalian
two-hybrid system
Firefly luciferase is a monomeric enzyme of
61kDa that catalyzes a two-step oxidation In conjunction with other techniques, the
reaction to yield light, usually in the green to mammalian two-hybrid system is used to
yellow region, typically 550–570nm. Upon characterize protein:protein interactions in a
mixing with substrates, firefly luciferase true mammalian environment. Due to the
produces an initial burst of light that is format, this technique is not usually used to
measured by a luminometer. The assay for screen large numbers of prey proteins.
firefly luciferase is easily quantitated and is
linear over at least seven orders of magnitude.
Primary reagent requirements for the
mammalian two-hybrid system
SEAP • Mammalian expression vector
SEAP is a protein that is secreted from cells containing DNA binding domain (e.g.,
and thus can be assayed using a small aliquot GAL4) upstream of coding sequences
of cell culture media. The enzyme can for the bait protein
withstand temperatures as high as 65ºC. • Mammalian expression vector
Therefore, endogenous alkaline phosphatase containing activation transcriptional
domain (e.g., VP16) and coding
activity can be eliminated by pretreatment of
sequences for the prey protein
the samples at 65°C. SEAP activity can be
• Appropriate mammalian cell line
measured using chemiluminescence or
fluorescence detection methods. • Cell culture media
• Transfection reagent
• Reagents for detection of reporter gene
β-Galactosidase output (e.g., luminometer, fluorometer)
Bacterial β-galactosidase consists of four
identical 116kDa subunits, and is extremely
stable and resistant to proteolytic
degradation. The most common method used
to detect
β-galactosidase activity is based on the ability
of the enzyme to hydrolyze ONPG to free o-
nityrophenol. The substrate
o-nityrophenol is yellow in aqueous solutions
and absorbs light at 420nm. Fluorescent,
chemiluminescent and colorimetric detection
methods are available. The fluorescent and
chemiluminescent methods are 20- to 1,000-
fold more sensitive than the standard
colorimetric method.
Chapter Three: Pull-down Assays

Contents

Pull-down assay formats

Page

Pull-down assays overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

3
CHAPTER

PULL-DOWN
GST pull-downs ASSAYS
Bacterial expression of both bait and prey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Bacterial expression of bait protein. Mammalian expression of prey protein . . . . . . . . . .8
Bacterial expression of bait protein. Mammalian cell-free expression of prey protein . . .9
HaloTag-based pull-downs
Mammalian cell expression of both bait and prey protein . . . . . . . . . . . . . . . . . . . . . . . .10
Mammalian cell-free expression of both bait and prey protein . . . . . . . . . . . . . . . . . . . .11
When to use pull-down assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Reagent requirements for GST-based pull-downs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Reagent requirements for HaloTag pull-down assays in mammalian cells . . . . . . . . . . . . . . . . . . . . . .12
Reagent requirements for HaloTag pull-down assays using
cell-free expression systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Pull-down assays overview

Pull-down assays probe interactions between a protein of interest that is expressed as a fusion
protein (e.g., bait) and the potential interacting partners (prey).
In a pull-down assay one protein partner is expressed as a fusion protein (e.g., bait protein) in
E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized
bait protein can then be incubated with the prey protein. The source of the prey protein
depends on whether the experiment is designed to confirm an interaction or to identify new
interactions. After a series of wash steps the entire complex can be eluted from the affinity
support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by
SDS-PAGE.
Successful interactions can be detected by Western blotting with specific antibodies to both the
prey and bait proteins, or measurement of radioactivity from a [35S] prey protein.

PROMEGA PROTEIN INTERACTION GUIDE 7

PULL-DOWN ASSAYS
REFERENCES
Bacterial expression of both bait
and prey proteins
1. Simader, H. et al. (2006) Nuc.
Acids. Res. 34, 3968–79.
2. Phillips, P. et al. (2006)
J. Biol. Chem. 281, 4903–10.
3. Reinert, L. (2006) Cancer. Res.
66, 8994–9001.
4. Onken, B. et al. (2006) Proc.
Natl. Acad. Sci. 103, 9045–50.
5. Li, H. et al. (2006) J. Biol.
Chem. 281, 14748–55.
Bacterial expression
of bait (GST fusions) and
mammalian cell expression
of prey
1. Osler, M. et al. (2005) J. Cell
Sci. 118, 4667–78.
2. Ingham, R. et al. (2005) Mol.
Cell. Biol. 25, 7092–106.
3. Hirst, J. et al. (2005) Mol. Biol.
of the Cell 16, 2554–65.
4. Wißmüller, S. et al. (2006) Nuc.
Acids Res. 34, 1735–44.
5. Chan, W. et al. (2005)
J. Biol. Chem. 25, 23741–47.
Bacterial expression of bait
(polyhistidine fusions),
mammalian cell expression of
prey
1. Vermeulen, M. et al. (2006) Mol.
Cell. Biol. 26, 5226–36.
2. Shao, H. et al. (2006)
J. Immunol. 176, 2933–41.
3. Hublitz, P. et al. (2005) Genes
and Development 19, 2912–24.
4. Desterro, J. et al. (2005) Mol.
Biol. Cell 16, 5115–26.
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Pull-down assay formats Bacterial expression of bait protein. Mammalian
expression of prey protein (Figure 3)
GST pull-downs
In this format the bait protein is expressed as
Bacterial expression of both bait and prey a GST fusion protein in E. coli followed by
proteins immobilization on particles containing reduced
The most commonly used method to glutathione.
generate a bait protein is expression as a Expression of endogenous or recombinant
fusion protein contain a GST (glutathione-S- prey proteins in eukaryotic cells increases the
transferase) tag in E. coli. This is followed by probability that they will be properly modified
immobilization on particles that contain or folded. These modifications can be critical
reduced glutathione which binds to the GST- for successful protein:protein interactions to
tag of the fusion protein. The primary occur.
advantage of a GST tag is that it can increase
the solubility of insoluble or semi-soluble Mammalian cell extracts containing the
expressed prey protein are prepared and
proteins expressed in E. coli.
allowed to interact with the immobilized GST
An alternative fusion tag such as 6X fusion bait protein.
polyhistidine can also be used to express the
prey or bait protein in E. coli. Using this
format, large amounts of bait and prey protein
partners can be expressed. Since both
proteins are expressed in a prokaryotic
environment, interactions that require folding
or modification of one or both partners may
not occur.
Bait Expressed
as GST Fusion
Endogenous Expressed in E.coli
Prey Protein from
Cell Lysis GST B Bait
Mammalian Cells
Bind/Wash/Capture on
Mammalian Glutathione Particles
Prey
P Cell
Glutathione
Particle GP GST B
Prepare Cell
Extract
Mix and Allow
Interaction to Occur
GP GST B P
Wash and Elute Complex
B P
6543MA
Gel Electrophoresis Followed
by Western Blotting
Figure 3. Schematic of pull-down assay using bacterial expression of bait protein and mammalian expression of prey protein.
The sequence of the bait protein is cloned into a vector containing a GST tag and the appropriate elements for growth and expression in E.
coli. Following expression the GST bait fusion protein is purified using glutathione particles, which bind to the GST tag. A cellular extract is
prepared from mammalian cells containing the prey protein. An aliquot of the cell extract is then allowed to interact with the bound GST bait
fusion protein for several hours. After washing away non-specifically bound proteins the complex is eluted by adding reduced glutathione
and analyzed by Western blotting.
Bacterial expression of bait protein. Mammalian
cell-free expression of prey protein (Figure 4)
Utilizing this format requires that the bait
protein be expressed as a (GST) fusion
protein in E. coli and immobilized on a
support resin. Mammalian, cell-free
using cell-free expression systems prey
proteins can be expressed in 1-2 hours and
used without purification. This convenience
enables the rapid characterization of several
different prey protein domains created by
site-directed mutagenesis. The effects of
specific mutations on the interaction can then
3
CHAPTER

PULL-DOWN
expression systems are used to generate a
be evaluated. ASSAYS
non-fusion prey protein. Expressed prey
proteins are then allowed to interact with the
immobilized GST fusion bait proteins. When

Bait expressed
as GST Fusion
Cell Free Expression in E.coli
of Prey Protein
GST B Bait

RNA Cell Lysis

Promoter Prey
P Prey Bind/Wash/Capture on
Sequence
Glutathione Particles REFERENCES (CONTINUED)
Bacterial expression of bait
Glutathione protein (GST fusions) mammalian
Particle GP GST B cell-free expression systems as
the source of prey protein
Plasmid Containing Mix and Allow
Protein Coding Sequence Interaction to Occur 1. Liang, S. and Lutz, C. (2006)
For Prey Protein RNA 12, 111–21.
2. Gu, X. et al. (2006) J. Bio.
GP GST B P Chem. 281, 17882–89.
3. Cohen, R. et al. (2006)
J. Clin. Endocrinol. Metab. 91,
Wash and Elute Complex
239–47.

B P 4. Hoberg, J. et al. (2006)

Mol. Cell. Biol. 26, 457–71.
HaloTag pull-downs using
6544MA

Gel Electrophoresis Followed

by Western Blotting mammalian cell-free expression
Figure 4. Schematic of pull-down assay using bacterial expression of bait protein and mammalian cell-free systems for the systems or mammalian cell
expression of prey protein. The sequence of the bait protein is cloned into a vector that contains the GST tag and the appropriate extracts
elements for growth and protein expression in E. coli. Following expression the GST bait fusion protein is immobilized using glutathione
particles which bind to the GST tag. The sequence of the prey protein is cloned into a vector containing the appropriate elements for 1. Urh, M. et al. (2005) Promega
expression using a mammalian-based cell-free expression system. An aliquot of the expressed prey protein is then allowed to interact with Notes 92, 21–4.
the bound GST bait fusion for several hours. After washing away non-specifically bound proteins the prey is eluted and analyzed typically by
2. Los, G.V. et al. (2005) Cell
Western blotting. Using an alternative procedure the prey may be expressed as [35S]-labeled protein and detected directly in the gel.
Notes 11, 2–6.

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PROMEGA PROTEIN INTERACTION GUIDE 9

PULL-DOWN ASSAYS

Pull-down assay formats Resin). This covalent linkage enables

HaloTag-based pull-downs
Mammalian cell expression of both bait and prey
proteins (Figure 5)
The properties of the HaloTag fusion protein
provide some important advantages that
increase the chance of successfully isolating
interacting partners. First the HaloTag protein
provides covalent attachment to a resin
containing a HaloTag ligand (e.g., HaloLink
extensive washing to remove nonspecifically
bound proteins, which can be problematic for
most pull-down experiments.
Using this format, cells are transfected with
HaloTag bait fusion protein and allowed to
form complexes with endogenously
expressed prey proteins. Cells are lysed and
the complexes captured by adding the
HaloLink Resin. After a series of washing
steps the prey are detected by gel analysis
followed by Western blotting.

Mammalian HaloTag Fusion

Cell Bait Protein
Endogenous
HaloTag B P Expressed
Prey Protein

HaloTag B P
Protein Interaction
Complex

Prepare
Cell Extract

HaloLink HLR HaloTag B P

Resin

Incubate With HaloLink

Resin and Capture Complex

HLR HaloTag B Wash and Elute Prey

P
6545MA

Gel Electrophoresis Followed

by Western Blotting
Figure 5. Schematic of HaloTag pull-down assay using mammalian cells to express both the prey and the bait proteins. The
protein coding sequence of the bait protein is cloned into a HaloTag vector containing the necessary elements for growth and protein
expression in mammalian cells. This recombinant vector is then transfected into the appropriate mammalian cell line. The HaloTag bait
fusion interacts and forms a complex with the prey protein. Whole cell extracts are then prepared. The complex is then immobilized using
the HaloLink Resin which forms a covalent bond with the HaloTag fusion tag. After extensive washing of the immobilized complex to remove
non-specific proteins, the prey is eluted and typically analyzed by Western blotting or mass spectrometry.

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10
Mammalian cell-free expression of both bait and
prey proteins (Figure 6)
Mammalian cell-free expression systems can
also be used to express both bait and prey
proteins. The use of GST or polyhistidine
fusion proteins can be problematic in cell-
protein is expressed as HaloTag fusion. The
protein complex is allowed to form and then
captured (using HaloLink Resin) and washed.
The prey is then eluted from the complex and
analyzed by gel electrophoresis.
3
CHAPTER

free expression systems because of high
background. These technical issues can be
resolved by expressing the bait protein as a
HaloTag fusion protein.
Using this approach, the prey protein is
expressed as a [35S] or fluorescent-labeled
protein containing no fusion tag. The bait
PULL-DOWN
When to use pull-down assays ASSAYS
Since pull-down assays can utilize prey
proteins expressed in several different ways,
such as endogenously in mammalian cells or
in cell-free expression systems this technique
can be used as either a discovery or a
characterization tool.

Cell Free Expression Cell Free Expression HaloTag

of Prey Protein Fusion Bait Protein

RNA HaloTag
Prey RNA
Promoter Bait
Sequence Promoter
Sequence
Prey

P HaloTag B

Plasmid Containing Plasmid Containing

Protein Coding Bait Coding Sequence
Sequence of Prey Mix and Allow
Complex to Form

HaloTag B P

HaloLink Add HaloLink Resin

HLR HaloTag B P and Capture Complex
Resin

Wash and Elute Prey

HLR HaloTag B

P
6546MA

Gel Electrophoresis Followed

by Western Blotting
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Figure 6. Schematic of pull-down assay using cell-free expression systems to express both bait and prey proteins. The protein-
coding sequences of the bait and prey proteins are cloned into individual vectors containing the necessary elements for expression using Phone
1-800-356-9526
mammalian-based cell-free systems. Proteins are expressed and aliquots from each reaction are mixed and the complex is allowed to form.
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The complex is then immobilized using the HaloLink Resin which forms a covalent bond with the HaloTag fusion tag. After extensive washing
1-800-356-1970
of the immobilized complex to remove non-specific proteins, the prey is eluted and typically analyzed by Western blotting. Using an alter-
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P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 11
PULL-DOWN ASSAYS

Reagent requirements Reagent requirements for HaloTag

for GST-based pull-downs
• Glutathione particles
• 2X SDS gel loading buffer
• SDS polyacrylamide gel
• Appropriate GST vector containing
coding sequences for bait protein
• Source of prey protein (e.g., cell-free
expression system, mammalian cells)
• Western blotting/antibodies (if using
non-labeled prey)
• [35S] methionine (if using labeled prey
proteins from a cell-free expression
system)
pull-down assays in mammalian cells
• Transfection reagent
• Mammalian cell line expressing prey
protein
• Appropriate HaloTag vector containing
coding sequences for bait protein
• HaloLink Resin
• Antibodies to prey and bait proteins
• Western blotting reagents
Reagent requirements for HaloTag
pull-down assays using cell-free expression
systems
• 2X SDS gel loading buffer
• SDS polyacrylamide gel
• Cell-free expression system
• Appropriate HaloTag vector containing
protein-coding sequences for the bait
protein
• Appropriate vector containing protein-
coding sequences for the prey protein
• HaloLink Resin
• Western blotting (if using non-labeled
prey)
• [35S] methionine (if using labeled prey)

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12
Chapter Four: Co-immunoprecipitation

Contents

Co-immunoprecipitation formats
Page

Overview of co-immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

4
CHAPTER

CO-
Mammalian cell-based formats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 IMMUNO-
P R E C I P I TAT I O N
Mammalian cell-free expression based formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
When to use co-immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Reagent requirements for mammalian cells as source of prey/bait proteins . . . . . . . . . . . . . . . . . . . .16
Reagent requirements for co-immunoprecipitation using
cell-free expression systems as source of prey/bait proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

Overview of co-immunoprecipitation
One of the most common and rigorous demonstrations of protein:protein interaction is the
co-immunoprecipitation of suspected complexes from cell extracts. Co-immunoprecipitation
confirms interactions utilizing a whole cell extract where proteins are present in their native
conformation in a complex mixture of cellular components that may be required for
successful interactions. In addition, use of eukaryotic cells enables post-translational
modification which may be required for interaction and which would not occur using
prokaryotic expression systems.
In a typical experiment cells are lysed and a whole cell extract is prepared under nondenaturing
conditions. It is critical to use non-denaturing conditions in order to maintain any interactions
that occur. An antibody specific to the bait is then added to the extract, forming a new
complex. This protein:protein complex is then immobilized on protein A or protein G sepharose
beads. Proteins that do not bind are removed by a series of washes. The protein complex is
then eluted from the beads and dissociated by SDS sample buffer. Samples are then evaluated
by SDS-PAGE followed by Western blotting with specific antibodies for the bait or prey
partners.

P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 13
CO-IMMUNOPRECIPITATION

Co-immunoprecipitation formats If antibodies are not available or endogenous

Mammalian cell-based formats
(Figure 7)
Mammalian cell-based formats allow
characterization of interacting partners using
endogenously expressed proteins. The
individual proteins should express well and
antibodies should bind under non-denaturing
conditions. This approach avoids the
overexpression of proteins that can occur
when using recombinant expression vectors.
It also allows detection of native sublocation
and post-translational modification, and
eliminates possible issues associated with use
of foreign linker and tag sequences.
levels of individual proteins are very low, then
recombinant vectors that contain short tags
and encode for the relevant proteins may be
transfected into the appropriate cell line.
These tags (e.g., Myc) are recognized by
commercially available monoclonal antibodies
and can be incorporated either at the
carboxyl or amino terminus of the bait or prey
proteins.

Mammalian
Cell

Endogenous Endogenous
Bait Protein B P Prey Protein or
or Expressed by Expressed by
Transfected Vector Transfected Vector
B P Protein Interaction
Complex Formed

Prepare Whole Cell

REFERENCES Extract and Add
Antibody to One of
Key original reference for the Protein Partners
co-immunoprecipitation B P

1. Phizicky, E.M. et al. (1995)

Y Antibody
Microbiol. Rev. 59, 94–123.
Capture Complex
Endogenously expressed bait and With Protein A/G-Sepharose
prey proteins in mammalian cells B P
Y
1. Schilders, G. et al. (2005) Nuc. Protein PA/G-S
Acids Res. 33, 6795–804. A/G-Sepharose
2. Napoli, A. et al. (2005) Nuc. Wash and
Acid Res. 33, 564–76. Elute Complex
3. McCracken, S. et al. (2005) J.
Biol. Chem. 280, 42227–36. B P
4. Miles, R. et al. (2005) Mol. Cell.
6547MA

Prot. 4, 1898–909. Gel Electrophoresis Followed

by Western Blotting

Figure 7. Schematic of immunoprecipitation from mammalian cells. Mammalian cells are cultured using conditions that stimulate the
endogenous expression and complex formation of prey and bait protein partners. A whole cell extract is prepared using conditions to
maintain the integrity of the complex. Antibodies to either partner are added and the complex is captured using protein A or protein G-
TO ORDER sepharose. After washing to remove non-specific proteins, the bait and prey proteins are eluted and analyzed by Western blotting using
Phone antibodies to either partner.
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14
Mammalian cell-free expression based formats
(Figure 8)
Mammalian cell-free expression systems may
also be used for co-immunoprecipitation. This
method avoids the need for transfection and
the generation of whole cell extracts, yet
As with cell-based experiments, complexes
form and appropriate antibodies to either the
bait or prey are added. This complex is then
immobilized on Sepharose beads that are
coupled to protein A or protein G. Non-
specifically bound proteins are washed away
from the complex. The partners are then CO-
4
CHAPTER

enables proteins to be expressed in a

analyzed by gel electrophoresis, followed by IMMUNO-
eukaryotic environment. Prey and bait
autoradiography or Western blotting. PRECIPITATION
partners can be expressed individually or
together in a single reaction. One of the
proteins can be labeled with [35S], fluorescent
tagged tRNAs or both partners can be
unlabeled.
REFERENCES (CONTINUED)
Recombinant vectors for
Cell Free Expression Cell Free Expression expression of bait and prey
of Prey Protein of Bait Protein
proteins in mammalian cells
RNA Prey RNA
Promoter Bait 1. Williams, T. et al. (2005) Nuc.
Promoter Sequence Sequence Acid Res. 33, 4475–84.
Prey Bait 2. Wilson, S. et al. (2005)
P J. Biol. Chem. 280, 28663–74.
B
3. Lussier, M.J. et al. (2005)
J. Biol. Chem. 280, 19393–400.
4. Shogomori, H. et al. (2005) J.
Plasmid Containing Plasmid Containing Biol. Chem. 280, 18931–42.
Protein Coding Protein Sequence
Mix and Allow 5. Forsthoefel, D. et al. (2005)
Sequence of Prey for Bait
Complex to Form Development 132, 1983–94.
6. Lechardeur, D. et al. (2005) J.
B P Biol. Chem. 280, 40216–25.

Add Antibody to Bait and prey proteins expressed

Prey or Bait Protein in cell-free expression systems
1. Yoshimi, R. et al. (2006)
Y Antibody
B P
J. Immunology 176, 3611–24.
Add Protein 2. Hu, P. et al. (2006) Mol. Cell.
A/G-Sepharose Biol. 26, 3071–84.
3. Belle-Poirier, M. et al. (2006) J.
B P
Y Mol. Endo. 36, 313–25.
Protein PA/G-S
A/G-Sepharose 4. Yu, Z. et al. (2005) Nucl. Acids
Res. 33, 1–12.
Wash and Elute Complex
5. Zissimopoulos, S. et al. (2005)
J. Biol. Chem. 280, 5475–85.
B P
6. Seo, S. et al. (2005)
Development 132, 105–15.
6548MA

Gel Electrophoresis Followed

by Western Blotting 7. Carson, C. et al. (2005)
Neuroscience 25, 6092–104.
Figure 8. Schematic of immunoprecipitation using cell-free expression systems. Coding sequences for the bait and prey proteins are
cloned into individual vectors which contain the necessary elements for expression in mammalian-based cell-free systems. Following
expression, aliquots from each reaction are mixed and the complex allowed to form. Antibodies to either partner are added and the complex TO ORDER
is then captured using protein A/G-sepharose. After washing to remove non-specific proteins, the bait and prey proteins are eluted and Phone
analyzed by Western blotting using antibodies to either partner. Using an alternative procedure, either the prey or bait may be expressed as 1-800-356-9526
[35S] or fluorescent-labeled protein and then detected directly in the gel. Fax
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P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 15
CO-IMMUNOPRECIPITATION

When to use co-immunoprecipitation Reagent requirements for

co-immunoprecipitation using
Since co-immunoprecipitation can utilize prey
cell-free expression systems as source of
and bait proteins expressed in several
prey/bait proteins
different ways, either endogenously in
mammalian cells or from cell-free expression • Cell-free expression system
systems, the technique can be utilized as • Cell-free expression vectors encoding
either a discovery or a characterization tool. sequences for both bait and prey
Use of cell-free systems for the expression of protein
both prey and bait proteins is ideal for the • [35S] methionine (if expressing labeled
rapid characterization of interactions using prey)
various mutant proteins to map domains • Sepharose Protein A or G
required for interactions. • Primary antibodies to either bait and
prey proteins (if using unlabeled prey)
• Western blotting reagents
Reagent requirements for mammalian cells as
source of prey/bait proteins
• Appropriate cell line that expresses
prey and bait proteins or mammalian
expression vectors expressing bait and
prey proteins
• Cell culture reagents
• Transfection reagent (if using recom-
binant vectors for expression)
• Sepharose Protein A or G
• Antibodies to bait and prey proteins or
antibody to tag present in the
expression vector
• Western blotting reagents

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16
Chapter Five: Co-localization/FRET/BRET

Contents

Co-localization assay formats

Page

Co-localization overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

5
CHAPTER

CO-
Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 L O CA L I Z AT I O N /
FRET/BRET
FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
When to use co-localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Reagent requirements for immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Reagent requirements for FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Reagent requirements for BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Co-localization overview
Methods such as co-immunoprecipitation and pull-downs require the preparation of cell
extracts which may not preserve the physiological conditions under which proteins may
interact in a true cellular environment. Using various co-localization techniques protein:protein
interactions may be characterized directly in the cell without the need to create cell lysates or
isolate complexes from a cell.

P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 17
CO-LOCALIZATION/FRET/BRET
REFERENCES
Key original reference for FRET
1. Gordon, G. et al. (1998)
Biophys. J. 74, 2702–13.
Key original reference for BRET
1. Xu, Y. et al. (1999) Proc. Natl.
Acad. Sci. 96, 151–56.
Use of immunofluorescence
1. Snabaitis, A. et al. (2006)
J. Biol. Chem. 281, 20252–62.
2. Vandermoere, F. et al. (2006) J.
Biol. Chem. 281, 14307–13.
3. Burgess, A. et al. (2006)
J. Gen. Virol. 87, 789–93.
Use of FRET
1. Sohn, H-W. et al. (2006) Proc.
Natl. Acad. Sci. 103, 8143–48.
2. Treanor, B. et al. (2006)
J. Cell. Biol. 174, 153–61.
3. Hunger, K. et al. (2006)
J. Bact. 188, 240–8.
4. Herrick-Davis, K. (2006)
J. Biol. Chem. 281, 27109–16.
5. Kramer, J. et al. (2006)
J. Immunol. 176, 711–15.
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18
Co-localization assay formats FRET
Immunofluorescence Fluorescence Resonance Energy Transfer
(FRET, Figure 9) can be used to measure
Immunofluorescence can be used to
interactions between two proteins in vivo.
determine whether two proteins share the
Using this technique, two different fluorescent
same location in a cell. Two primary
molecules (donor and acceptor fluorophores)
antibodies that recognize the two specific
are used to label the suspected protein
proteins are added simultaneously to the
partners. FRET is observed by exciting the
sample. Two secondary antibodies with
sample at the donor excitation wavelength
different fluorescent tags are then added. The
and measuring fluorescence intensities
sample is then analyzed under a confocal
emitted at the wavelengths corresponding to
microscope to determine whether the two
the emission peaks of the donor compared to
fluorescent signals overlap. If both fluorescent
those of the acceptor. When the donor and
signals are in the same location then the
acceptor are in close proximity (1-10nm) due
proteins are located in the same cellular
to the interaction of the two proteins, the
region. This is an indirect way of determining
acceptor emission is predominantly observed
whether two proteins may interact; if they are
because of the intermolecular FRET from the
located in the same region there is a
donor to the acceptor.
possibility that they may bind to each other.
Another option to labeling proteins with
When available, immunofluorescent antibodies
fluorescent dyes is to express both partners
to endogenously expressed proteins should
as fusion proteins with different GFP (green
be used. However, when antibodies are not
fluorescent protein) tags. The most popular
available, or when the cell line does not
FRET pair for biological use is a cyan
express the protein at sufficient levels for
fluorescent protein (CFP)-yellow fluorescent
immunofluorescence detection, cells may be
protein (YFP) pair. Both are color variants of
transfected with recombinant vectors
GFP.
encoding tagged proteins. Antibodies to the
tag can then be used to localize the protein in
the cell.
Excitation
458nm
FRET
P
CF
A Energy Transfer
B 480–525nm
YF
6640MA
P
Emission
525-575nm
Figure 9. Overview of FRET for the analysis of protein:protein interactions. Protein partner A is cloned into a vector containing cyan
fluorescent protein (CFP, donor). The second partner B is cloned into a vector containing a yellow fluorescent protein (YFP, acceptor). The
vectors should also contain the appropriate elements for protein expression in mammalian cells. Both recombinant vectors are then trans-
fected into mammalian cells. FRET can be observed by exciting the sample at the donor excitation wavelength while measuring
fluorescence intensities emitted at the wavelengths corresponding to the emission peaks of the donor compared to that of the acceptor.
When the donor and acceptor are in close proximity, acceptor emission is predominantly observed because of the intermolecular FRET from
the donor to the acceptor.
BRET
Bioluminescence Resonance Energy Transfer
(BRET) involves the transfer of energy from a
donor enzyme to suitable acceptor molecule.
Using this method one protein partner is
expressed as a fusion with GFP and the other
Reagent requirements for immunofluorescence
• Confocal laser scanning microscope
(and appropriate filters)
• Tissue culture equipment
• Mammalian cells
• Transfection reagents CO-
5
CHAPTER

is expressed as fusion with Renilla. BRET • Fluorescently labeled antibodies LOCALIZATION/

technology is based on the transfer of
resonant energy from a bioluminescent donor
protein to a fluorescent acceptor protein using
Renilla luciferase (R luc) as the donor and a
GFP mutant as the acceptor molecule. BRET
technology is analogous to FRET, but
eliminates the need for an excitation light
source and its associated problems such as
high background caused by
autofluorescence.
When to use co-localization
Co-localization is used in conjunction with
other techniques to characterize
protein:protein interactions in a true
mammalian environment. This technique is
typically not used for screening large numbers
of prey proteins.
FRET/BRET
Reagent requirements for FRET
• Confocal laser scanning microscope
(and appropriate filters)
• Tissue culture equipment
• Mammalian cells
• Transfection reagents
• Fluorescent dyes
• Mammalian expression vectors
containing donor tag (e.g., CFP) and
coding sequences for one protein
partner
• Mammalian expression vectors
containing acceptor tag (e.g., YFP) and
coding sequences for the other protein
partner
Reagent requirements for BRET
• Microplate reader capable of
luminescent and fluorescent detection
• Tissue culture equipment
• Mammalian cells REFERENCES (CONTINUED)
• Transfection reagents Use of BRET
• Mammalian expression vectors 1. Shimizu, T. et al. (2006) Pro.
containing donor tag (e.g., Renilla) and Natl. Acad. Sci. 103, 2093–97.
coding sequences for one protein
2. Whittard, J. et al. (2006)
partner
Mol. Biol. Cell 17, 2696–706.
• Mammalian expression vectors
3. Goin, J. et al. (2006) J. Biol.
containing acceptor tag (e.g., YFP) and
Chem. 281, 5416–25.
coding sequences for the other protein
partner 4. Rebois, R-V. et al. (2006)
J. Cell. Sci. 119, 2807–18.
5. Koshimizu, T. et al. (2006) Mol.
Pharmol. 69, 1588–98.
6. Sirokmany, G. et al. (2006)
J. Biol. Chem. 281, 6096–105.

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P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 19
 6
CHAPTER

CHROMATIN
Chapter Six: Chromatin immunoprecipitation (ChIP)

Contents

Chromatin immunoprecipitation overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

Chromatin immunoprecipitation formats
Page

IMMUNO- Antibody format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

PRECIPITATION
Antibody-free format; Use of HaloTag fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
When to use chromatin immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Primary reagent requirements for chromatin immunoprecipitation assays . . . . . . . . . . . . . . . . . . . . .23

Chromatin immunoprecipitation overview

Chromatin immunoprecipitation (ChIP) is an experimental method used to determine whether
proteins, such as certain transcription factors, are associated with a specific genomic region in
living cells or tissues. This cell-based technique is often used together with non-cell-based
assays to characterize protein:DNA interactions.
The method is based on the principle that formaldehyde reacts with primary amines located on
amino acids and the bases on DNA or RNA molecules, forming a covalent crosslink between
the specific protein to the DNA on which they are situated.
Following crosslinking, the cells are lysed and the crude cell extracts are sonicated to shear the
DNA to a smaller size. The protein:DNA complex is immunoprecipitated using an antibody
against the protein of interest. The DNA protein cross-links are reversed by heating and the
proteins removed by treatment with proteinase K. The DNA portion of the complex is then
purified and identified by PCR using specific primers to the suspected binding region.

20
Chromatin immunoprecipitation
(ChIP) formats
Antibody format
The classic format of the ChIP assay follows
the basic procedure noted in Figure 10 and
requires four days for completion. The
to the protein of interest. If antibodies are not
available the proteins can be fused to tags
such as HA or c-myc, which are recognized
by commercially available antibodies. The
success of the procedure relies on the ability
of the antibody to bind to the target protein
after crosslinking (crosslinking changes
6
CHAPTER

CHROMATIN
epitope recognition of the antibody). IMMUNO-
procedure requires highly specific antibodies PRECIPITATION

Transcription Factor
TF

Crosslink with Formaldehyde

TF TF Lysis of Cytoplasm

Nuclei Lysis in 1% SDS

Experimental Control
Sonicate
TF TF

Split Sample
Antibody
Incubate +/- Antibody
O/N at 4°C
TF TF REFERENCES
Incubate with Protein A/G Key original reference for
Agarose 2 hours at 4°C chromatin immunoprecipitation
A/G 1. Solomon, M. et al. (1985) Proc.
Wash
TF Natl. Acad. Sci. 82, 6470–74.
A/G TF
Elute with TE +1% SDS Antibody format
1. Benson, L. et al. (2006)
Proteinase K, Reverse J. Biol. Chem. 281, 9287–96.
Crosslinks—O/N at 65°C
TF TF 2. Ghosh, M. et al. (2006) Mol.
Cell. Biol. 26, 5270–83.
Purify DNA
6641MA

3. White, D. et al. (2006) Cancer

Res. 66, 3463–70.
4. Lei, H. et al. (2006) Proc. Natl.
Figure 10. Overview of chromatin immunoprecipitation using antibodies. Mammalian cells are grown using the appropriate condi-
tions to modulate the formation of protein:DNA interactions. To conserve the DNA:protein structure during cell lysis formaldehyde is added Acad. Sci. 103, 10305–09.
resulting in the formation of cross-links between the DNA and the bound protein. A whole-cell extract is prepared, and the cross-linked 5. Shur, I. et al. (2006) Stem Cells
chromatin is sheared by sonication to reduce average DNA fragment size. Either a polyclonal or monoclonal antibody to the target protein is 24, 1288–93.
added. The success of the procedure relies on the use of an antibody that will specifically and tightly bind its target protein under the buffer
and wash conditions used. Protein A/G agarose beads are added to capture the complex and incubated overnight. Reversal of the 6. Natesampillai, S. et al. (2006) J.
formaldehyde cross-linking by heating permits the recovery and quantitative analysis of the immunoprecipitated DNA. Biol. Chem. 281, 3040–47.

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P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 21
CHROMATIN IMMUNOPRECIPITATION

Antibody-free format; use of HaloTag fusion of chromatin, lysed and then sonicated. Then,
proteins (Figure 11) instead of using an antibody to capture the
DNA:protein complex, the complex is
An assay format that does not require the use
captured directly onto the HaloLink Resin. The
of antibodies is based on the HaloTag
HaloLink Resin provides a method for
Technology. A HaloTag vector containing the
covalent, oriented attachment of HaloTag
protein-coding sequence of interest is
fusion proteins onto a solid surface. The
transfected into the appropriate mammalian
DNA:protein crosslinks are reversed by
cell line. The cells are then fixed with
heating, and the DNA can then be purified
formaldehyde, allowing the HaloTag fusion
using commercially available columns.
protein to be crosslinked to the specific region

Covalent Capture of Chromatin Complexes

Using HaloTag® Technology

Transfection
HT

HaloTag®
TF
Expression of HaloTag®
Vector TF
Fusion Protein

HT HT

TF Formaldehyde Cross-linking
HaloCHIP™
Blocking Ligand
Experimental Lysis, Sonication
Control
Sample Sample

1–1.5 days
HaloLink™
HT HaloLink™ HT Split Sample
Add HaloCHIP™ Blocking
TF TF Ligand to the control
sample to prevent
binding to HaloLink™ Resin

Capture on HaloLink™ Resin

HaloLink™
HT HaloLink™
Wash HaloLink™ Resin
TF Covalent capture allows for
highly stringent washes
REFERENCES (CONTINUED) to remove non-specific
proteins and DNA
Genome-wide chromatin
immunoprecipitation assays
Release of DNA by reversal
1. Krieg, A. et al. (2006) Mol. Cell. of cross-links
Biol. 26, 7030–45. Sample DNA Background DNA

2. Mayanil, C. et al. (2006)

J. Biol. Chem. 281, 24544–52.
3. Kajiyama, Y. et al. (2006) Analyze Analyze
J. Biol. Chem. 281, 30122–31.
Figure 11. Capture of protein:chromatin interactions using HaloTag technology. The protein-coding sequence of a transcription factor
(TF) is cloned into a HaloTag (HT) vector containing the necessary elements for protein expression in mammalian cells. This recombinant
vector is transfected into mammalian cells. Mammalian cells are then grown under the appropriate conditions to modulate the formation of
protein:DNA interactions. In order to conserve the DNA:protein structure formaldehyde is added resulting in the formation of cross-links
between the DNA and the bound protein. A whole-cell extract is prepared, and the cross-linked chromatin is sheared by sonication to
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When to use chromatin immunoprecipitation
Chromatin immunoprecipitation can be used
to confirm the location of individual or multiple
transcription factors during cell growth or
upon exposure to abnormal conditions such
as UV radiation. The technique can also be
Primary reagent requirements for chromatin
immunoprecipitation assays
•
•
•
•
Mammalian cell line of choice
Cell culture media
Polyclonal or monoclonal antibody
Cell lysis reagent
6
CHAPTER

CHROMATIN
utilized in conjunction with microarrays to • Formaldehyde IMMUNO-
discover the location of various transcription • Protein A/G agarose PRECIPITATION
factors on a genome-wide basis.
• Proteinase K
• DNA purification reagents
• PCR reagents (including primers
specific for the DNA region of interest)
• HaloTag fusion protein (specific for
HaloTag procedure)
• HaloLink Resin (specific for HaloTag
procedure)

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 7
CHAPTER

ELECTRO-
Chapter Seven: Electrophoretic mobility shift assay (EMSA)

Contents Page

EMSA overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

EMSA assay formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
PHORETIC When to use EMSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
MOBILITY
SHIFT ASSAY Primary reagent requirements for EMSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

Electrophoretic mobility shift assay overview

The Electrophoretic Mobility Shift Assay (EMSA) also referred to as the gel retardation assay or
gel shift assay, is a common technique used to characterize protein:DNA/RNA interactions. Gel
shift assays are often performed concurrently with DNase footprinting, ChIP and primer
extension assays. EMSA/gel assay is based on the observation that complexes of protein and
DNA/RNA migrate through a nondenaturing polyacrylamide gel more slowly than free
DNA/RNA fragments or double-stranded oligonucleotides. The gel shift assay is performed by
incubating a purified protein, or a complex mixture of proteins with a 32P biotinylated labeled or
hapten end-labeled DNA/RNA fragment containing the putative protein binding site and
nonspecific DNA competitors usually polyanion polymers such as poly(dI-dC) or poly(dG-dC).
These repetitive polymers provide an excess of nonspecific sites to adsorb proteins in crude
lysates that will bind to any DNA or RNA sequence. The complex is then analyzed on a
nondenaturing polyacrylamide gel or TAE agarose gel. The ability to resolve protein:DNA/RNA
complexes depends largely upon the stability of the complex during its migration into the gel.
Sequence-specific interactions are transient and are stabilized by the relatively low ionic
strength of the electrophoresis buffer used.
The specificity of an observed DNA/RNA binding reaction can be evaluated using competition
assays in which an excess of unlabeled probe is added together with the labeled probe.
Specific DNA or RNA binding will be eliminated by a reasonable excess (10–100 molar excess)
of unlabeled probe (unlabeled specific competitor).
In addition to a labeled DNA fragment, specific antibodies can be added to the gel shift
reaction. Addition of a specific antibody to a binding reaction can have one of several effects. If
the protein recognized by the antibody is not involved in complex formation, addition of the
antibody should have no effect. If the protein that forms the complex is recognized by the
antibody, the antibody can either block complex formation, or it can form an antibody-protein-
DNA complex, resulting in a further reduction in the mobility of the protein-DNA complex
(supershift).

24
EMSA assay formats (Figure 12)
Target proteins may be obtained from crude
cellular extracts or cell-free expression
systems, or may be purified from E. coli or
mammalian expression systems. Cellular
extracts are easy to prepare and allow
proteins may play a critical role. DNA/RNA
binding proteins generated by cell-free
expression systems or by purification from
other expression systems offer an excellent
format for the confirmation of data obtained
from cellular extracts, and for characterization
of the context of the protein:DNA interactions.
7
CHAPTER

ELECTRO-
protein:DNA/RNA interactions to occur in a PHORETIC
true cellular environment in which other MOBILITY SHIFT
ASSAY
Cell-Free Expression Endogenous Expressed
of Protein Protein
RNA Protein of Mammalian
Promoter Interest Cell REFERENCES
P Key original reference for EMSA
1. Garner, M. et al. (1981) Nuc.
Acids. Res. 9, 3047–60.
or Target protein obtained from
mammalian cellular extracts
1. Liu, F. et al. (2006) Mol. Cell.
P Biol. 17, 585–97.
Incubate With 2. Kim, E. et al. (2006)
Labeled Double J. Immunol. 176, 256–64.
Stranded DNA Oligo
3. Huang, C. et al. (2006)
32P J. Immunol. 176, 4173–81.
Run Samples on
Non-Denaturing 4. Khanna, H. et al. (2006)
gel Polyacrylamide Gel J. Biol. Chem. 281, 27327–34.

32P
5. Trujillo, M. et al. (2006) Mol.
P Positive Interaction Endocrinol. 20, 2559–75.
(shift in mobility)
Target protein obtained from
Negative Interaction
6554MA

32P cell-free expression systems

(no shift)
1. Brunner, C. et al. (2006) Nuc.
Figure 12. Overview of EMSA. The target protein is expressed in mammalian cells and a whole-cell extract is prepared. An alternate Acids Res. 34, 1807–15.
method relies on expressing the protein using mammalian-based, cell-free expression systems. In either case an aliquot containing the 2. Yasuhiko, Y. et al. (2006) Proc.
protein is incubated with the DNA sequence that has been labeled using either radioactive or non-radioactive methods and the DNA:protein Natl. Acad. 103, 3651–56.
complex is allowed to form. In order to maintain the protein:DNA complex, the reaction is run on a non-denaturing polyacrylamide gel. After
electrophoresis, the experimental reaction is compared to a control reaction that contains only the labeled DNA to determine whether a 3. Huang, J. et al. (2006)
protein:DNA interaction has occurred. J. Virol. 80, 1098–09.
4. Bose, F. et al. (2006) Mol. Cell.
Biol. 26, 3942–54.
5. Akasaka, T. et al. (2006) Proc.
Natl. Acad. Sci. 103,
11999–04.

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P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 25
ELECTROPHORETIC MOBILITY SHIFT ASSAY

When to use EMSA Primary reagent requirements for EMSA

EMSA can be used in conjunction with other • Neutral non-denaturing polyacrylamide
techniques to characterize the transcriptional gel
regulation mechanism of various genes. It can • Labeled and unlabeled DNA/RNA
also be used to further characterize known fragment containing putative binding
transcriptional events to determine the effect site
REFERENCES (CONTINUED) of various stimuli or other upstream proteins • Target protein (from cellular extract,
that are required for successful interactions. expressed in cell-free system or purified
Target purified proteins from from mammalian or E. coli expression
E. coli (Polyhistidine- systems)
or GST-tagged) • Poly (dI-dC) or other nucleic acids to
1. Wen, Y. et al. (2006) J. Bact. lower non-specific background
188,1750–61.
2. Ding, X. et al. (2006) Nucl.
Acids Res. 34, 2570–78.
3. Kawai, S. et al. (2006) Genes
Cells 11,163–75.
4. Sikorski, E. et al. (2006)
J. Biol. Chem. 281, 24423–30.
5. Croteau, D. et al. (2006)
J. Biol. Chem. 281, 26370–81.
Supershift assays
1. Liu, G. et al. (2006) J. Biol.
Chem. 281, 29479–90.
2. Song, C. et al. (2006) Mol.
Endocrinol. 20, 795–808.
3. Dinur, M. et al. (2006) Mol.
Endocrinol. 20, 1652–60.
4. Mickleburgh, I. et al. (2006)
RNA 12, 1397–1407.
5. Arai, K. et al. (2006) Mol.
Cancer. Res. 4, 247–55.
Protein:RNA interactions
1. Gorrill, T. et al. (2006)
J. Gen. Virol. 87, 1557–66.
2. Richter, S. et al. (2006) Nuc.
Acids Res. 34, 4278–92.
3. Schultz, A. et al. (2006)
J. Biol. Chem. 281, 28278–86.
4. Clerte, C. et al. (2006) RNA 12,
457–75.
5. Yarovinsky, T. et al. (2006)
J. Immunol. 177, 4426–35.

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26
Chapter Eight: Appendix

Contents Page

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
8
CHAPTER

APPENDIX

P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 27
APPENDIX
Glossary
Common terms used in proteomics research
BRET (Bioluminescence Resonance
Energy Transfer): This technology is based
on the transfer of resonant energy from a
bioluminescent donor protein to a fluorescent
acceptor protein using Renilla luciferase
(Rluc) as the donor and a mutant of the Green
Fluorescent Protein (GFP) as the acceptor
molecule.
Cell-free expression: Cellular extract
containing all the components required for the
coupled transcription/translation of protein-
coding DNA sequences.
Co-immunoprecipation: An experiment
designed to affinity purify a bait protein
antigen together with its binding partner using
a specific antibody against the bait.
Denature: To cause a protein to fold or unfold
into a structure other than it’s native 3-D
conformation.
Endoplasmic reticulum (ER): A membrane
system that extends throughout the
cytoplasm and is involved in the synthesis,
processing, transport, and secretion of
proteins.
EMSA: EMSA (Electrophoretic Mobility Shift
Assay), also known as gel retardation/gel shift,
the assay is based on the fact that
protein:DNA complexes migrate more slowly
through a native polyacrylamide or agarose
gel than unbound DNA. The individual
protein:DNA complexes can be visualized as
discreet bands within the gel using
chemiluminescence or radioisotopic
detection.
Expression profiling: A high-throughput
method for evaluating the degree and timing
of gene expression in a cell or tissue.
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FRET: FRET (Flourescence Resonance
Energy Transfer) is a technique that can
measure interactions between two proteins in
vivo. The occurrence of FRET can be
observed by exciting the sample at the donor
excitation wavelength while measuring
fluorescence intensities emitted at the
wavelengths corresponding to the emission
peaks of the donor compared to those of the
acceptor. Donor emission intensity decreases
while acceptor intensity increases. When the
donor and acceptor are in close proximity,
FRET occurs and the acceptor emission is
observed.
Functional proteomics: The large-scale
study of protein function, especially
protein:protein interaction networks,
biochemical pathways, and post-translational
modifications.
GST (glutathione-S-transferase): A 26kDa
fusion tag developed from Schistosoma
japonicum that has a strong affinity for
glutathione covered matrices. GST-fusion
protein binding to glutathione is reversible,
allowing efficient elution of the bound GST-
fusion protein by addition of reduced
glutathione to the elution buffer.
Glycoprotein: A protein with covalently
bound carbohydrates.
Glycosylation: Post-translational addition of
carbohydrate groups to a molecule.
Glycosylation of proteins occurs via the amide
group within the sequence Asn-X-Ser/Thr (or
through the hydroxyl of the serine or threonine
residue in the sequence).
HaloTag Interchangeable Labeling
Technology: A novel tool for imaging live or
fixed mammalian cells that express the
HaloTag protein or protein fusions, for
analyzing post-translational modification of
labeled fusion proteins, and for isolating
proteins and protein complexes. The
technology is based on the efficient formation
of a covalent bond between a specially
designed ligand and the protein encoded by
the HaloTag gene.
Heat-shock protein: A protein synthesized in
response to cellular stress, including high
temperature. Heat-shock proteins function as
molecular chaperones to protect proteins
from mis-folding.
Kozak sequence: A DNA sequence that
surrounds the ATG start signal for the
translation of an mRNA.
Mass spectrometer (MS): An instrument
that determines the exact mass of charged
particles or ions by measuring the flight path
through a set of magnetic and electric fields.
Mass spectrometers specialized for protein
and peptide sequencing are used for high-
throughput identification.
Nuclear magnetic resonance (NMR): A
spectroscopic technique used to determine
the 3-D structure of small- to medium-sized
proteins. NMR is based on resonant
absorption of electromagnetic radiation by the
magnetic dipole moments of atomic nuclei in
an applied magnetic field.
Open Reading Frame (ORF): The DNA
sequence between the translation start signal
and the termination codon that can be
translated into a protein.
Peptide: Two or more amino acids joined by
a peptide bond.
Peptide bond: An amide bond formed
between two amino acids by the linkage of
the amino group of one amino acid to the
carboxyl group of a second amino acid.
Peptide map, peptide fingerprint: A pattern
produced by hydrolysis of a protein and 2-D
mapping of the resulting peptide fragments.
Phage display (peptide phage display): A
technique that fuses peptides to capsid
proteins on phage surface. Libraries of phage-
displayed peptides may be screened for
binding to specific ligands; determination of
the gene sequence of the selected phage
identifies the peptide sequence.
Polyhistidine-tag: Consists of approximately
six histidine residues near the N- or C-
terminus of a protein. The total number of
histidine residues may vary. Polyhistidine tag
fusion proteins bind to nickel and other metals
and are eluted by the use of imidazole.
P R O M E G A P R O T E I N I N T E R A C T I O N G U I D E 29
Post-translational modification:
Modification of proteins following translation,
including glycosylation, phosphorylation,
sulfation, acetylation, and ribosylation.
Prenylation: The addition of a prenyl moiety
to a protein. The addition of prenyl groups
8
CHAPTER
APPENDIX
regulates protein-membrane interactions.
Primary antibody: An antibody generated
against an antigenic target (a protein, peptide,
carbohydrate, or other small molecule).
Protease: An enzyme that degrades proteins
by hydrolyzing peptide bonds.
Proteasome: A large protein complex that
degrades proteins that have been tagged for
elimination, particularly those tagged by
ubiquitination.
Protein domain: A structurally and
functionally defined protein region. In proteins
with multiple domains, the combination of the
domains determines the function of the
protein.
Protein fingerprint: The pattern of proteins
in a cell or organism as determined by 2-D gel
electrophoresis.
Proteome: The dynamic protein complement
of an organism, including all post-translational
modifications and protein interactions.
Pull-down Assay: An affinity
chromatography method that involves using a
tagged or labeled bait to create a specific
affinity matrix that will enable binding and
purification of a prey protein from a lysate
sample or other protein-containing mixture.
Riboproteomics: The systematic
characterization of RNA:protein interactions
that affect the splicing, transport, lifetime and
translation of RNAs.
Secondary antibody: An antibody that
recognizes and binds a primary antibody.
Secondary antibodies conjugated to enzymes
and labels are key components of detection
systems.
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APPENDIX

Signal peptidase: An endopeptidase that Ubiquitin: A protein that is covalently

removes the signal peptide (signal sequence) attached to lysine residues of other proteins,
following translocation of a protein. tagging them for proteolysis within
proteasomes. Multiple ubiquitin units may be
Signal sequence: A short amino acid
ligated to the protein, forming a multi-ubiquitin
sequence that determines the localization of a
chain.
protein within the cell.
Western blot: A technique for the separation,
Structural proteomics: Large scale
immobilization, and detection of proteins,
determination of protein structures in three-
usually by a labeled antibody. Proteins are
dimensional space. Common methods are x-
separated by gel electrophoresis, transferred
ray crystallography and NMR spectroscopy.
to a membrane, and then probed with
Two-dimensional electrophoresis (2-D antibodies, which are then detected by
gel): A technique used for the separation of chemiluminescent or colorimetric methods.
complex protein mixtures. Proteins are
separated in the first dimension on an
isoelectric focusing gel, then separated by
molecular weight using standard gel
electrophoresis.

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