Innovative Solutions For Fluorescent and Chemiluminescent Applications - BioFiles Issue 4.1

Life Science
BioFiles
Volume 4, Number 1
Innovative Solutions for Fluorescent and

Chemiluminescent Applications
DNA Detection
New NIR Fluorescence Dyes
Carbohydrate Detection
Green fluorescent protein (GFP) in a
Pacific jellyfish, Aequorea victoria
Protein Detection
Isoelectric Focusing
Detection Kits
Sensor Dyes
Life Science
BioFiles
Volume 4, Number 1
Your gateway to Biochemicals and Reagents

for Life Science Research from Sigma® Table of Contents
Introduction 3
DNA Detection 4
Nancy-520 for Robust
DNA Staining and Quantitation 4
DNA and RNA Intercalating Fluorophores 5
New NIR Fluorescence Dyes 6

Tracy 645 and Tracy 652 6
Carbohydrate Detection 10
BioFiles Online allows you to:
New Fluorescently Labeled Lectins 10
• Access any issue of BioFiles
Fluorescent Labels for Carbohydrates 11
• Subscribe for:
✓ future issues Protein Detection 12
✓ eBioFiles email notifications LUCY® Protein Stains 12
• Stay up-to-date on the latest BioNews from Sigma
Isoelectric Focusing 14
Register today for upcoming issues and eBioFiles announcements and receive
a free IUBMB-Sigma-Nicholson Metabolic Pathway Chart: sigma.com/biofiles Fluorescent IEF-Markers 15
Buffers for Capillary Zone Electrophoresis 16
Detection Web Resource Detection Kits 17

Ampliflu Red Western Blot Kit 17
The detection web resource includes selection tables and learning center
Nitrite/Nitrate Assay Kit 18
pages for a large number of luminescent probes and indicators, stains and
dyes, microparticles, embedding media, and substrates.
Sensor Dyes 19
The detection resource also contains two useful tools for designing and Fluorogenic NO Bioimaging Probes 19
implementing a detection technique that is right for your research. Chelators and Ion Probes 22
Method-to-Product Guide pH Indicators 23
Enables users to select the correct product based on the method of detection Liphophilic and Membrane Probes 24
being used or vice versa.
New Product Corner 26
For example, the method-to-product guide can help select the correct stains
and labels for detecting proteins Books 27
by flow cytometry or the correct
probes for lipophilic and membrane
norm. fluorescence
detection by microscopy.
Wavelength Index
Authors of articles:
Quickly find excitation and emission Alex Rueck, Ph.D.; Monika Baeumle, Ph.D.;
Bernhard Schoenenberger, Ph.D.
wavelengths for a large number of
550 600 650 700 750
detection products. wavelength (nm) Other technical contribution:
Klaus Trummler, Ph.D.; Jakob Zbären, Inselspital Bern,
Detection products and web tools can be found at sigma.com/fluorescence Switzerland
2
Introduction
Monika Baeumle, Ph.D.
Product Manager, Biochemistry
monika.baeumle@sial.com
The 2008 Nobel Prize for Chemistry was This issue of BioFiles focuses on these new products for
awarded to Osamu Shimomura, Martin fluorescent bioanalytical techniques
Chalfie and Roger Tsien for the discovery
Nancy-520 for DNA staining
and development of the green fluorescent
protein, GFP. GFP was first isolated from Fluorescent near-IR dyes Tracy 645 and Tracy 652
Introduction
the jellyfish, Aequorea victoria in 1962 by Atto-dye conjugated lectins for carbohydrate analysis
Shimomura. Since then, it has become one of the most important
LUCY® dyes for enhanced protein staining
tools used in life science research. GFP enables researchers to
investigate processes within living cells. This award emphasizes Fluorescent isoelectric focusing (IEF) markers
the importance of luminescent techniques as one of the most Ampliflu Red Western Blot Kit
important, widespread and fast-growing analytical tools in life
Highly sensitive Fluorogenic NO Bioimaging Probes for
science and analytical biochemistry.
intracellular measurements
Fundamental requirements in fluorescent techniques are We hope that you will find our articles and products to be
high sensitivity and selectivity, allowing a scientist to track interesting and helpful. For further information, please visit our
molecular interactions, mobility, and conformational changes of detection web resource at sigma.com/fluorescence.
biomolecules. Because of our extensive experience in organic
synthesis, a tradition of highest quality assurance and our focus
on analytical techniques for chemical and biochemical research, Single molecule studies are one
Sigma-Aldrich® has been a leading supplier of fluorescent of the hottest topics in biological
probes for many years. In order to ensure the highest level of sciences because they bring us closer
performance, we continuously investigate and develop new to understanding cellular processes.
products and optimize application protocols for the fields of Read and comment on this exciting
detection and biochemistry. Our Detection Center of Excellence application of detection research at
located in Buchs, Switzerland has risen to this challenge and has the BioBlog.
developed a series of novel fluorescent dyes.
Our Innovation, Your Research — Shaping the Future of Life Science 3

DNA Detection
Nancy-520 for Robust DNA Staining and Quantitation
Nancy-520 is a fluorescent stain for double stranded DNA
(dsDNA) on agarose electrophoresis gels, with higher sensitivity
than ethidium bromide and an easy, fast, and robust staining
procedure. It is less mutagenic than Ethidium bromide and SYBR®
Green I, according to Ames test. Nancy-520 has an excitation 23130 –
maximum at 520 nm and an emission maximum at 560 nm 9416 –
6557 –
DNA Detection
(see Figure 1). Nancy-520 is provided as a 5000× stock solution 4361 –

(500 μl), which is sufficient for 50 agarose gels. The limit of
2322 –
detection is 0.5 ng/band of dsDNA. In addition, Nancy-520 can be 2027 –
used to determine dsDNA concentrations in solution.
Nancy-520 + dsDNA in TBE, pH 8.3

1.2 564 –
1
0.8
0.6
Figure 2. DNA marker (Lambda DNA Hind III digest) at 2 different concentrations, was
0.4 separated on a 1% agarose gel, stained with Nancy-520 post-electrophoresis and imaged
under 2 different conditions. Left: λex UV-Screen (300 nm)/λem 590 nm bandpass filter/CCD
0.2
camera. Right: Laser-scanner Fuji FLA-3000/λex 532 nm/λem 580 nm cut-off filter.
0
400 450 500 550 600 650 700 DNA Quantitation in Solution
nm
Nancy-520 can be used to determine dsDNA concentrations in
Figure 1. Normalized fluorescence excitation (red) and emission (blue) spectra of
Nancy-520 in the presence of dsDNA, measured in a cuvette on a Varian Cary® Eclipse
solution. This application can be performed in a glass-bottomed
fluorescence spectrophotometer with 2 mL of TBE, pH 8.3. The excitation spectrum 96 well plate, using known concentrations of dsDNA as a
was measured at a fixed emission wavelength of 560 nm, and the emission spectrum standard. Nancy-520 has a linear detection range between
was measured at a fixed excitation wavelength of 520 nm.
0-2 μg/ml of DNA (see Figure 3).
DNA Staining 5000
Nancy-520 can be used in a standard post-electrophoresis staining 4500

4000
application for DNA on agarose gels. After the electrophoresis run,
3500
the gel is stained for 1 hour in the dark. The fluorescence image of
3000
the gel can be directly detected after a short rinse (see Figure 2).
F/u
2500
Alternatively, Nancy-520 can also be used for pre-electrophoresis 2000
staining by simply adding the dye to the liquid agarose before 1500
pouring the gel. The gel can be imaged directly after the 1000
electrophoresis run without any further steps. Nancy-520 allows 500
more detection possibilities than SYBR® Green. Detection is 0

0 500 1000 1500 2000 2500
performed by illuminating the gel on a UV screen or using a Dark
ng/ml DNA
Reader®, and imaging the gel using a CCD-camera (e.g., KODAK®
Figure 3. Quantitation of DNA (Lambda DNA Pst I Digest) in solution using Nancy-520,
Gel-Logic-100) with a 535 nm or 590 nm band-pass filter, or a performed in a 96 well plate. Fluorescence was measured on a laser-scanner (FLA-3000,
Polaroid® camera. Alternatively, a laser-scanner can be used Fuji) with 532 nm excitation and 580 nm emission filter. Different concentrations of DNA
in TE buffer, pH 7.5, and their corresponding fluorescence values are shown.
(e.g., Fuji® FLA-3000) with a 473 nm excitation setting and
520 nm emission filter, or with a 532 nm excitation setting and
580 nm emission-filter. Other imaging systems are possible with
similar excitation sources and emission filter settings.
4 sigma.com/lifescience Order: sigma.com/order Technical service: sigma.com/techinfo

Nancy-520 and Related Products for DNA Analysis
Description Cat. No.
Nancy-520 01494-500UL
GenElute™ Agarose Spin Columns 56500-70EA
Agarose A4679-50G
A4679-100G
A4679-500G
Gel Loading Buffer G2526-5ML
Lambda DNA Hind III Digest D9780-20UG

D9780-.1MG
D9780-.5MG
D9780-1MG
DNA Detection
Lambda DNA Pst I Digest D1793-.1MG
D1793-.5MG
D1793-1MG
Tris-Borate-EDTA buffer T7527-1L

T7527-4L
Tris Acetate-EDTA buffer T8280-100ML

T8280-1L
DNA and RNA Intercalating Fluorophores

Nucleic acid research requires the highest sensitivity. Fluorescent labeling, as well as radioisotopic labeling, have found wide application,
and fluorescence techniques in particular allow real time analysis with high resolution. Furthermore, fluorescence labeling techniques
eliminate the hazards and waste disposal restrictions of radioisotopes.
There are different approaches to fluorescently labeling DNA or RNA. A common strategy is the use of intercalators, small molecules that are
incorporated into the DNA molecule. This incorporation produces a dramatic increase in fluorescence of the intercalators.
Description λex/λem (nm) Cat. No.
Acridine Orange hemi(zinc chloride) salt - 158550-10G

158550-25G
158550-100G
Atto-Dino 2 solution - 83399-200UL-F
Atto-Dino 4 solution - 83392-200UL-F
Atto-Mono 1 - 67888-250UL-F
Atto-Mono 2 - 51796-250UL-F
bisBenzimide H 33258 355 / 465 nm in TE buffer; DNA 14530-100MG

14530-500MG
bisBenzimide H 33342 trihydrochloride 355 / 465 nm in TE buffer; DNA 14533-100MG
4′,6-Diamidino-2-phenylindole dihydrochloride 374 / 461 nm in 10 mM Tris; 1 mM EDTA; pH 8.0; DNA 32670-5MG-F

32670-25MG-F
Dimidium bromide 306 / 612 nm in 50 mM Tris pH 8.0 (DNA) 41785-250MG

41785-1G
Ethidium bromide 530 / 600 nm in 50 mM phosphate buffer pH 7.0 46065-1G

(upon binding to DNA) 46065-5G
Ethidium bromide solution 530 / 600 nm in 50 mM phosphate buffer pH 7.0 46067-50ML-F

(upon binding to DNA) 46067-250ML-F
Ethidium homodimer 528 / 598 nm in TE buffer; DNA 46043-1MG-F

46043-5MG-F
5-Methoxypsoralen - 275727-250MG
275727-1G
Propidium iodide - P4170-10MG

P4170-25MG
P4170-100MG
P4170-250MG
P4170-500MG
P4170-1G
Propidium iodide solution - P4864-10ML
Pyrene 338 / 375 nm in DMSO 82648-1G

82648-10G
SYBR® Green II RNA gel stain - S9305-.5ML

S9305-1ML

Tracy 645 and Tracy 652
An increasing demand for red emitting fluorescent dyes, useful Tracy 652 and Tracy 645 surpass conventional fluorescent labels,
in life science applications such as antibody and protein labeling, such as Cy® 5, in all of the above criteria (see Figure 1). Other,
prompted us to develop new improved long wavelength markers more modern labels of the corresponding type are also deficient in
for use in the visible/near-infrared (NIR) region. one or more of the above criteria.
Tracy 645 and Tracy 652 (see Table 1) are proprietary dyes 900
developed to meet the need for NIR fluorescent dyes. The Tracy
dyes are:
fluorescence intensity [au]

Able to retain chemical stability of the activated 800
N-hydroxysuccinimide (NHS) ester under protein labeling
Tracy 652 (Cat. No. 38408)
conditions (basic pH, which prevents dye decomposition or Cy5 (Amersham)
hydrolysis that could result in poor labeling efficiency).
700
Photostable.
Soluble in aqueous labeling media as the activated NHS ester,
reducing precipitation that leads to inefficient protein labeling.
600
0 5 10 15
Flexible for use in typical protein labeling protocols.
time of irradiation [h]
The decrease in background fluorescence results from both a Figure 1. Photostability of Tracy 652 dye and Cy® 5.
dramatic reduction of Rayleigh and Raman scattering by shifting
Irradiation of each 1mM solutions in methanol in a white Pyrex flask by a 375 W
to the longer wavelength excitation and from the diminished white light lamp; distance from lamp to probe was 20 cm; probe maintained at room
autofluorescence of sample impurities in this region. temperature; measured at λex,max and λem,max of each dye.
Investigation of optimal dye/protein (D/P) ratio of antibody

Tracy 645 (free acid) Tracy 652 (free acid) conjugates of Tracy dyes showed a maximum emission intensity
λ λex, max (639 nm) 280 nm λex, max (648 nm) 280 nm at ~4:1 D/P.
absorption 1.818
0.343 1 μg/mL 0.495 50 μg/mL 0.373 0.455 50 μg/mL Secondary antibody conjugates with Tracy dyes are available for
(PBS, pH 7) μg/mL
correction
0.029 0.044 immunostaining applications observed by fluorescence microscopy
factor[4]
(see Figure 2).
ε 222882 M-1cm-1 6437 M-1cm-1 181253 M-1cm-1 8028 M-1cm-1
658nm (1 μg/mL, 0.1M phosphate 672nm (1 μg/mL, 0.1M phosphate
λem,max
buffer pH 7) buffer pH 7)
Table 1. Spectroscopic data of Tracy fluorophores, useful for dye/protein ratio

calculation of conjugates.
Several major advantages of the red emitting fluorescent dyes over

conventional shorter wavelength ones make the Tracy dyes highly
attractive for cellular and molecular biology or bioanalytical studies.1-3
Reduced background improves sensitivity.
Strong fluorescent signal of labeled proteins at various
dye/protein ratios.
Low-cost, dependable laser diodes may be used as excitation
sources instead of short-lived, expensive gas lasers. Figure 2. Confocal microscopic image (CLSM) of a rat stomach section after
immunohistological staining (2-channel mode). Green channel: staining of cytokeratin.
Primary antibody: anti-cytokeratin from rabbit; secondary antibody: anti-rabbit IgG
labeled with Atto 488 dye (λex 488 nm). Red channel: Staining of actin. Primary
antibody: anti-actin from mouse; secondary antibody: anti-mouse IgG labeled with
Tracy 645 dye (λex 543 nm).

Additionally NTA (nitrilo-triacetate)-Tracy derivatives that selectively 500 250 100 50 5
Marker
detect His-tagged proteins on solid surfaces such as gels or [ng]
5 50 100 250 500
Western blots are provided by Sigma® Life Science (see Figure 3).
His-Ladder
500 ng
250 ng
100 ng
50 ng
25 ng
100–
Protein 1: Atto550
45– Protein 2: Tracy652
30–
75 —
50 — His-p38 12–

30 —
Figure 4. Immunoblot detection of Protein 1 and Protein 2 using two primary
antibodies and two anti-IgG dye conjugates. Sequential imaging with a FLA-3000
Fuij® laser scanner. First image for Atto 550: λex 532 nm with 580 nm emission filter.
Second image for Tracy 652: λex 633 nm with 675 nm emission filter.
References
Figure 3. His-tagged p38-MAPK protein (500 ng–25 ng) was separated on a 4-20%
1. Wolfbeis, O.S., Fluorescence Spectroscopy: New Methods and Applications, Springer,
Tris-Glycine SDS-PAGE gel. The gel was fixed overnight in 40% ethanol/10% acetic
Heidelberg, (1993).
acid, washed in water, and incubated with Ni-NTA-Tracy 652 (1:1000) in the dark. The 2. Rettig, W. and B. Strehmel, B., Applied Fluorescence in Chemistry, Biology and
gel was washed and then imaged using a FLA-3000 Fuji® laser scanner with 633 nm Medicine, Springer, Heidelberg, (1999).
excitation and a 675 nm emission filter. Ni-NTA-Tracy 652 (λex 652 nm, λem 677 nm) is 3. Mason, W.T. (ed.), Fluorescent and Luminescent Probes for Biological Activity, 2nd
excited in the red region of the spectrum. ed., Academic Press, (1999).
4. Calculation of the dye/protein ratio [D/P or DOL] and the protein concentration
[cprotein] of labeled protein is according to Mujumdar, R.B., et al., Bioconj. Chem.,
The key advantages of this His-tagged protein detection method 4, 105 (1993).
as compared to classical chemiluminescence-based detection on
Western blots are the simplicity and efficiency of the protocol, but
at the cost of some sensitivity.
Tracy 652 has been applied in two-color multiplex immunoblotting:
Protein 1 and 2 in amounts ranging from 5 to 500 ng were A Perfect Fit!
separated on a 4-20% Tris-Glycine SDS-PAGE gel and then
transferred to a low-fluorescence PVDF membrane. The membrane
was blocked with 5% BSA in PBS, incubated with antibodies to
protein 1 and 2 respectively, and probed with the corresponding
secondary antibodies conjugated to fluorescent dyes Atto 550
(green) and Tracy 652 (red) (see Figure 4).
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Tracy Dyes Tracy-NTA Conjugates
Tracy 645 (activated) 8 NTA -Tracy 645 8
corresponds for coupling to amines Tracy 645- Nitrilotriacetic acid, complexed to Ni2+ - ion;
42445-1MG 1 mg Nα, Nα-bis(carboxymethyl)-L-lysine Nickel(II) complex - Tracy 645 conjugate
18626-250UG 250 μg
Tracy 645 (free acid) 8
NTA -Tracy 652 8
 ≥95.0% (HPCE)
12670-1MG 1 mg Tracy 652 - Nitrilotriacetic acid, complexed to Ni - ion;
2+
Nα, Nα-bis(carboxymethyl)-L-lysine Nickel(II) complex - Tracy 652 conjugate

Tracy 652 (activated) 8
 ≥95% (HPCE)
 ≥80% (coupling to amines) 55183-250UG 250 μg
42454-1MG 1 mg
Tracy 652 (free acid) 8 Tracy Protein Labeling Kits

 ≥95% (HPCE) Tracy Protein Labeling Kits provide an easy and reliable way to label
38408-1MG 1 mg purified proteins, enzymes and antibodies. The kits include a high
quality Tracy dye and contain all components necessary to rapidly
Tracy-Conjugated Antibodies label and purify five protein samples. A final dye/protein ratio [D/P or
DOL] of 2-9 can be expected, depending on the nature of protein.
Anti-Mouse IgG - Tracy 652 from goat antiserum 8
T
racy 652 - goat-Anti-mouse IgG Tracy 645 Protein Labeling Kit 8
1 mg/mL protein This kit contains sufficient amounts of reactive dye, buffers, and protein
free of unconjugated dye; dye-to-protein ratio ≥2 purification sets for performing five labeling reactions (1 mg protein each)
and for the subsequent purification of the labeled protein.
42472-1G 1 g
91471-1KT 1 kit
Anti--Rabbit IgG - Tracy 645 from goat antiserum 8
Tracy 652 Protein Labeling Kit 8
T
racy 645- goat-Anti--rabbit IgG
This kit contains sufficient amounts of reactive dye, buffers, and protein
1 mg/mL
purification sets for performing five labeling reactions (1 mg protein each)
free of unconjugated dye; dye-to-protein ratio >2 and for the subsequent purification of the labeled protein.
05557-1G 1 g
75824-1KT 1 kit
Anti-Rabbit IgG - Tracy 652 from goat antiserum 8
T
racy 652 - goat-anti-rabbit IgG
1 mg/mL protein
free of unconjugated dye; dye-to-protein ratio (molar) >2
43413-1G 1 g
Detection Web Resource
Over 1,000 Products for Find the Optimal Probe or Label!

Sensitive and Simple Detection Sortable wavelength index — search by
n Stains, Dyes, & Markers excitation or emission wavelength
n Fluorescent Micro- & Nanoparticles Method-to-product guide — find suitable
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New Fluorescently Labeled Lectins
Sigma® Life Science now offers a new series of Atto-dye labeled
lectins. Lectins are ubiquitous proteins or glycoproteins that can
be isolated from plant and animal sources and can bind to specific
carbohydrate moieties. These highly specific interactions play an
important role in many recognition processes in biological systems.
The recently developed Atto-dye labeled lectins are designed for
many detection applications, including carbohydrate, histochemical

and mitogenic studies. Atto-dyes have very bright fluorescent
signals with narrow fluorescence spectra and high photostability.
An extensive series of lectins and lectin conjugates is
available from Sigma Life Science. Learn more by visiting Fluorescence image of rat testis. Seminiferous tubule with maturing germ cells.
sigma.com/enzymeexplorer The acrosomal caps of the spermatids are specifically marked green (λex 485 nm)
(Lectin from Phytolacca americana-Atto 488 conjugate, Cat. No. 39905). The nuclei
are labeled blue with DAPI. Image by J. Zbären, Inselspital Bern, Switzerland.
Concanavalin A-Atto 488 conjugate 501 / 523 nm in PBS 40634-1MG
Concanavalin A-Atto 565 conjugate 563 / 592 nm in PBS 69535-1MG
Lectin from Phaseolus vulgaris-Atto 488 conjugate 501 / 523 nm in PBS 75319-1MG
Lectin from Phaseolus vulgaris-Atto 550 conjugate 554 / 576 nm in PBS 90852-1MG
Lectin from Phaseolus vulgaris-Atto 647N conjugate 644 / 669 nm in PBS 77363-1MG
Lectin from Phytolacca americana-Atto 488 conjugate 501 / 523 nm in PBS 39905-1MG
Lectin from Phytolacca americana-Atto 550 conjugate 554 / 576 nm in PBS 94816-1MG
Russia
SIGMA-ALDRICH RUS, LLC
Glycobiology
Lectin from Phytolacca americana-Atto 647N conjugate 644 / 669 nm in PBS 03065-1MG
Tel: +7 (495) 621 6037
Glycobiology Analysis Manual
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Lectin from Triticus vulgaris-Atto 488 conjugate 501 / 523 nm in PBS 16441-1MG
Analysis Manual
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■ Glycan Labeling and Analysis
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Detection
Whether you are an expert in carbohydrate biology and chemistry or just getting started in glycomics, the
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Glycobiology Analysis Manual provides the products and methods you need to solve your glycomics puzzle!
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Fax: (+358) 9 350 92555 Fax: (+39) 02 3801 0737 Fax: (+351) 21 924 2610 ■ Glycan Labeling and Analysis
■ Glycoprotein Purification and

Detection
■ Chemical and Enzymatic

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and its affiliate Sigma-Aldrich Biotechnology, L.P. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information
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Please see reverse side of the invoice or packing slip.
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GlycoProfile is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.
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Fluorescent Labels for Carbohydrates
Due to their various biological functions carbohydrates are experiencing a rapidly growing interest. Fluorescence labeling and detection of
carbohydrates enable highly sensitive investigation by requiring only small sample sizes. Depending on the carbohydrate-bearing moiety
(e.g. reducing functionality), different labeling approaches are required.
2-Aminoacridone 420 / 538 nm in 0.1 M Tris pH 8.0 06627-25MG

06627-100MG
7-Amino-4-methylcoumarin 365 / 440 nm in ethanol A9891-250MG

A9891-500MG
A9891-1G
A9891-5G
8-Aminonaphthalene-1,3,6-trisulfonic acid disodium salt 356 / 512 nm in 0.1 M phosphate pH 7.0 08658-500MG
9-Anthracenecarbonyl cyanide 361 / 451 nm (after derivatization with ethanol) 10609-25MG-F

10609-100MG-F
9-Anthracenecarboxaldehyde - 278688-5G
278688-25G
278688-100G
Bis(2,4-dinitrophenyl) oxalate / 430 nm 75705-1G

75705-5G
Bis(2,4,6-trichlorophenyl) oxalate / 430 nm 75707-5G

75707-25G
4-Bromomethyl-7-methoxycoumarin 322 / 395 nm (after derivatization) 235202-250MG

235202-1G
235202-5G
9-(Chloromethyl)anthracene 327 / 412 nm (after derivatization with sodium acetate) 25102-5G-F
4-Chloro-7-nitrobenzofurazan 420 / 540 nm in ethanol (after derivatization with glycine) 25455-1G

25455-5G
25455-25G
Dansyl chloride 337 / 492 nm in chloroform (after derivatization with hexylamine) 39220-1G-F
39220-5G-F
39220-50G-F
Dansylhydrazine 340 / 520 nm in ethanol 30434-250MG

30434-1G
30434-5G
7-(Diethylamino)coumarin-3-carbonyl azide 390 / 478 nm in methanol 31755-25MG
Diphenylborinic anhydride 366 / 475 nm (after derivatization) 358835-250MG

358835-1G
9-Fluorenylmethyl carbazate 264 / 309 nm (aldose adduct) 46917-250MG-F
Fluorescein-5-thiosemicarbazide 492 / 516 nm in 0.1 M Tris pH 9.0 46985-100MG-F

46985-500MG-F
4-Fluoro-7-nitrobenzofurazan 470 / 550 nm 47140-10MG

47140-50MG
4-Hydrazino-7-nitrobenzofurazan 445 / 522 nm in chloroform (after derivatization with formaldehyde) 53892-50MG
Isoniazid 360 / 450 nm (thiol adduct) I3377-5G

I3377-50G
I3377-250G
Malonamide 367 / 445 nm (α-keto acid adduct) 129593-100G
4-Methoxybenzamidine 393 / 478 nm (after derivatization with glucose) 64785-100MG-F

393 / 478 nm
4-(6-Methyl-2-benzothiazolyl)phenyl isocyanate 327 / 382 nm in methylene chloride (after derivatization with 65877-100MG
hexylamine) 65877-500MG
4,5-Methylenedioxy-1,2-phenylenediamine dihydrochloride 367 / 445 nm in 0.1 M phosphate pH 7.0 (after derivatization 66807-10MG
with pyruvate) 66807-50MG
1-Naphthaleneacetic anhydride 216 / 360 nm (polyamin adduct) 438952-250MG

438952-1G
1-(2-Naphthoyl)imidazole 234 / 374 nm (after derivatization) 70684-500MG
o-Phenylenediamine 337 / 417 nm (after derivatization) P5412-50TAB

P5412-100TAB
o-Phenylenediamine - 78410-100G
2-Phenylphenol - P28263-500G
P28263-2KG

Protein Detection
LUCY® Protein Stains
LUCY 506, LUCY 565, and LUCY 569 are fluorescence dyes
Marker
250
100
for the sensitive detection of proteins on 1- or 2-dimensional
75
50
25
10
1
electrophoresis gels. They are compatible with subsequent
MS-analyses (see Figure 4), have a broad linear range for
quantification, provide easy handling, and offer a quick staining
procedure. They are also available in a LUCY Starter Kit, containing 66 —
Protein Detection
66 —
all 3 dyes. In addition, protein quantification in solution can be
performed using LUCY 506 and LUCY 569. 45 —
45 —
1.20
29 —
1.00 29 —
0.80
0.60
0.40
Marker
0.20
Marker
250
100
250
100
75
50
25
10
75
50
25
10
1
5
0.00
220 270 320 370 420 470 520 570 620 670 720
[nm]
Figure 1. Normalized fluorescence excitation and emission spectra of the LUCY dyes
in the presence of BSA and SDS. 66 —
66 —
LUCY 506: Blue line: excitation spectrum (maximum 506 nm); pink line: emission
spectrum (maximum 520 nm). 45 —
45 —
LUCY 565: Orange line: excitation spectrum (maximum 565 nm); green line:
emission spectrum (maximum 588 nm).
29 —
LUCY
29 — 569: Yellow line: excitation spectrum (maximum 569 nm); light blue line:
emission spectrum (maximum 585 nm).
Protein Staining
LUCY 506 shows highest sensitivity, with a detection limit of
~ 3 ng protein per band. The standard procedure is a post-
electrophoretic stain with omission of the fixation step. The Figure 2. Mixture of 3 proteins (bovine serum albumin, ovalbumin, and carbonic
anhydrase) loaded in each lane (1-250 ng/band). Top: 10-20% Tris-glycine gel,
stain is completed after 60 minutes, and detection is possible
stained with LUCY 506. Bottom: 4-20% Tris-glycine gel, stained with LUCY 565.
immediately thereafter. Both gels were imaged on a FLA-3000 laser scanner (Fuji®).
LUCY 565 is recommended if after staining, a Western blot Depending on the chosen dye (see Figure 1), several devices can be
transfer is to be performed from the same gel. Since staining is used for the detection, e.g., illuminating the gel on a Dark-Reader®
done under neutral, non-fixing conditions, the stained proteins blue light transilluminator or a UV screen, and imaging the gel
may be transferred directly to the membrane. Using traditional using a CCD- or Polaroid® camera. Alternatively, a laser-scanner can
dyes, 2 gels and, therefore, twice the amount of sample be employed, using the corresponding excitation and emission filter
material would be needed. settings (see Figures 2 and 3).
LUCY 569 is particularly well-suited for protein quantification
in-gel. It has an extraordinary large linear dynamic range
between 10-6000 ng/band, which is a larger range than for
most silver staining methods, Coomassie® Brillant Blue, or other
fluorescence dyes.
Figure 3. Staining of 2D-minigels: 10 μg E. coli extract, 7 cm IPG-strips pH 3-10,

4-20 % Tris-Glycine gels (Imaging by laser scanner FLA-3000).
Left: LUCY 506 Right: SYPRO® Ruby.

% Int LUCY 506 solution 8
100
90 Lucy 506 is a fluorescent stain for protein electrophoresis, with high
80 sensitivity and easy, fast and robust staining procedure for all kinds of
70 SDS gels. Protein staining by Lucy 506 does not interfere with subsequent
60 MALDI-MS.
50 Sensitivity of routine procedure is ca. 3 ng/band, with linear response
40 between 3 and 1000 ng/band
30
Lucy 506 is provided as a 5000 x stock-solution in DMF (5 mg/ml)
20
10 λmax. ~495 nm (MeOH)
0 68721-500UL 500 μL
1000 1500 2000 2500 3000 3500 4000
m/z LUCY 565 solution
Figure 4. MALDI peptide mass fingerprint after in-gel-digest. 100 ng of E. coli
Protein Detection
 BioChemika
β-galactosidase, separated by SDS-PAGE, was stained with LUCY 506 after band Lucy 565 is a fluorescent stain for protein electrophoresis, with high
excision and trypsin-digest. MALDI spectra of extracted peptides, crystallized with HCCA sensitivity and easy, fast and robust staining procedure for all kinds of
and measured on a Shimadzu® Kratos CFR MALDI-MS instrument in reflectron mode. SDS gels. Lucy 565 is also suitable for neutral staining (e.g. for subsequent
western blotting).
Protein Quantification in Solution Protein staining by Lucy 565 does not interfere with subsequent MALDI-MS.
LUCY® 506 and LUCY 569 can be also used to quantify proteins Sensitivity of routine procedure is ca. 5 ng/band, with linear response
between 5 and 4000 ng/band
in solution. This application is designed for use in a 96-well format
λmax. ~553 nm (MeOH)
or in cuvettes. It is applicable for 2 different protein concentration
Lucy 565 is provided as a 5000 x stock-solution in DMSO (5 mg/ml)
ranges, 0-50 μg/ml and 10-1000 μg/ml and requires only buffer,
43772-500UL-F 500 μL
SDS, and the LUCY dye (see Figure 5). Samples can be measured
immediately after mixing. LUCY 569 solution
1. Low Range  BioChemika 2. High Range

12000 Lucy10000
569 is a fluorescent stain for protein electrophoresis, with good
sensitivity and easy, fast and robust staining procedure for all kinds of
10000 SDS gels.
8000
Lucy 569 provides a broad linear dynamic range for protein
quantification in electrophoresis. Protein staining by Lucy 569 does not
8000 interfere with subsequent MALDI-MS.
6000
Sensitivity of routine procedure is ca. 5 ng/band, with linear response
F/u
F/u
6000
between 5 and 6000 ng/band
4000
4000 λmax. ~559 nm (MeOH)
2000 Lucy 2000
569 is provided as a 5000 x stock-solution in DMSO (5 mg/ml)
41629-500UL-F 500 μL
0 0
0 10 20 30 40 50 60 LUCY Starter
0 Kit 200 400 600 800 10008
µg/ml BSA The LUCY Starter Kit contains three fluorescent
µg/ml BSAprotein stains for
SDS-PAGE gels.
2. High Range
L UCY 506 for high sensitivity staining with a detection limit of 3 ng
10000
protein per band. Staining is complete after 60 minutes and detection is
possible immediately thereafter.
8000
L UCY 565 is recommended for staining SDS-PAGE gels prior to Western
blot transfer. With traditional staining, 2 separate gels are required and
6000 therefore, double the amount of sample is necessary.
F/u
L UCY 569 is particularly well suited for protein quantitation, displaying a

4000
broad, linear, dynamic range between 10–6,000 ng protein per band.
Components
2000
LUCY 506 (Sigma 68721) 50 μL
LUCY 565 (Fluka 43772) 50 μL
0
60 0 200 400 600 800 1000
LUCY 569 (Fluka 41629) 50 μL
51153-1KT 1 kit
µg/ml BSA
Laemmli Lysis-buffer 8
Figure 5. Different concentrations of BSA in solution were quantified in the
96-well plate format using LUCY 506. Low range (Top) and high range (Bottom).  non smelling
Fluorescence detection was done on Fuji® FLA-3000 (λex 473 nm/λem 520 nm). in accordance for electrophoresis test
38733-1ML 1 mL
For more information about LUCY dyes and their applications in
38733-5X2ML 5 × 2 mL
protein analysis, visit sigma.com/lucy_dyes 38733-5ML 5 mL

Isoelectric focusing (IEF) is a powerful analytical tool for the To overcome these problems, Sigma® Life Science introduces
separation of proteins. In order to ensure the high performance of synthetic IEF-markers, which can be used for fluorescent detection.
analysis, isoelectric point (pI) standards are needed. In addition to The fluorescent IEF-markers enable a much higher sensitivity
classical protein based standards, low molecular weight compounds than using UV detection, as is commonly used with slab gel
have been developed and successfully examined in capillary IEF electrophoresis. The maximum absorbances of the individual
and IEF-gel electrophoresis. Gel electrophoresis, the common markers are between 308 and 350 nm. For fluorescence detection,
technology for IEF, minimizes convection and introduces an an excitation wavelength of 310 nm is suggested (individual
additional gel-sieving effect to separate proteins by size. However, excitation maxima range between 310 to 400 nm); the emission
it has several disadvantages such as lengthy analysis time, limited maxima of the individual markers are between 410 and 500 nm
resolution, and difficulty in detection. These challenges have been (see Figure 1). An advantage in IEF gel electrophoresis with
largely overcome by the development of IPG-strips (immobilized pH fluorescent IEF-markers is the ability to control the formation of a
gradients) for use in high-resolution pI-separation. gradient without further staining (using UV illumination).
Capillary electrophoresis is a high-resolution approach to separate Peak Number
molecules, based on the pI point, that can be automated. Separation 0.026
is carried out in fused-silica capillaries with internal diameters of
2
25-75 μm. The electrophoresis takes place in free solution and the
1
0.022
4
capillary controls convection currents. After focusing is complete,
3
the solutes are pumped out of the capillary. UV absorption is the 0.018
7
most popular detection method for capillary IEF, but UV induced
fluorescence emission is of interest for derivatized proteins. Dansyl 0.014
chloride, fluorescamine, o-phthaldialdehyde, and coumarin moieties A
u
are used to increase detection sensitivity. 0.010
One of the most critical issues in isoelectric focusing, whether in

0.006
capillary electrophoresis or slab gel, is standardization of results.
5
For that purpose typical protein standards have been used which
0.002
6
could show certain disadvantages:
Limited stability of solutions -0.002
Inadequate purity for application as a standard

-0.006
Lot-to-lot inconsistency
10 15
Minutes
Figure 1. CE-IEF with different fluorescent pI Markers: 1, pI 10.2; 2, pI 9.0; 3,
pI 7.6: 4, pI 7.2; 5, pI 5.5; 6, pI 4.0; 7, pI 3.0.
Reference
Horká, M., Willimann, T., Blum, M., Nording, P., Friedl, Z., Slaisk, K. Capillary isoelectric
focusing with UV-induced fluorescence detection, J. Chromatogr. A, 916, 65-71 (2001).

Fluorescent IEF-Markers
Fluorescent IEF-Marker pI 2.1 solution 340 / 430 nm in 50 mM citrate, 50 mM KCl, pH 2.1 74169-200UL-F
Fluorescent IEF-Marker pI 3.0 solution 360 / 440 nm in 0.1 M citrate pH 3.0 72172-200UL-F
Fluorescent IEF-Marker pI 4.0 solution 310 / 415 nm in 0.1 M citrate pH 4.0 89827-200UL
Fluorescent IEF-Marker pI 6.2 solution 394 / 505 nm in 0.1 M phosphate pH 6.2 73938-1EA
73938-200UL
Fluorescent IEF-Marker pI 6.6 solution 396 / 500 nm in 0.1 M phosphate pH 6.6 73376-1EA
73376-200UL
Fluorescent IEF-Marker pI 6.8 solution 338 / 418 nm in 0.1 M phosphate pH 6.8 89508-200UL-F
Fluorescent IEF-Marker pI 7.2 solution 387 / 500 nm in 0.1 M phosphate pH 7.2 89951-200UL-F
Fluorescent IEF-Marker pI 7.6 solution 385 / 495 nm in 0.1 M phosphate pH 7.6 89952-200UL
Fluorescent IEF-Marker pI 8.1 solution 340 / 420 nm in 0.1 M Tris pH 8.1 75734-200UL-F
Fluorescent IEF-Marker pI 8.7 solution 390 / 500 nm in 0.1 M Tris pH 8.7 89357-200UL
Fluorescent IEF-Marker pI 9.0 solution 385 / 495 nm in 0.1 M Tris pH 9.0 90699-200UL
Fluorescent IEF-Marker pI 9.5 solution 325 / 415 nm in 0.1 M carbonate pH 9.5 89268-200UL
Fluorescent IEF-Marker pI 10.3 solution 388 / 495 nm in 0.1 M carbonate pH 10.3 77672-200UL
IEF-Marker 3.6-9.3 - 56733-1EA-F
IEF mix 3.6-9.3 - I3018-1VL
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Chemistry and Biology are converging to transform
the study of cellular physiology and mechanisms of disease.
The new Bioactive Small Molecules catalog is your resource for the latest products
targeting the interface of Chemistry and Biology. Browse thousands of antiproliferatives,
enzyme inhibitors, GPCR ligands, and many other high quality small molecule probes.
Request your copy today at sigma.com/cellbiocat
sigma-aldrich.com

Buffers for Capillary Zone Electrophoresis
For your convenience—buffers for HPCE
We offer a complete range of ready-to-use buffers for high performance capillary electrophoresis. These buffers cover the pH range from
2.5 to 11 and meet the quality requirements for HPCE.
Description Cat. No. Description Cat. No.
Buffer solution HPCE pH 2.5, BioChemika, 82635-50ML Buffer solution HPCE pH 7.5, BioChemika, 82592-50ML
for HPCE, 50 mM sodium phosphate 82635-100ML for HPCE, 20 mM sodium phosphate 82592-100ML
82592-500ML
Buffer solution HPCE pH 2.5, BioChemika, 82578-50ML
for HPCE, 100 mM sodium phosphate 82578-100ML Buffer solution HPCE pH 7.7, BioChemika, 82619-50ML
82578-500ML for HPCE, for the detection of inorganic and 82619-100ML

low molecular weight organic anions
for HPCE, for the detection of alkali and alkaline 82621-100ML Buffer solution HPCE pH 8.0, BioChemika, 82594-50ML
earth metals and amines for HPCE, 20 mM sodium tetraborate 82594-100ML
for HPCE, 20 mM sodium citrate 82582-100ML for HPCE, 100 mM sodium borate 82634-500ML
for HPCE, 150 mM potassium phosphate 82622-500ML for HPCE, 50 mM sodium borate 82633-100ML
for HPCE, 20 mM sodium citrate 82583-100ML for HPCE, 20 mM sodium phosphate 82593-100ML
for HPCE, 20 mM sodium citrate 82584-100ML for HPCE, 20 mM sodium tetraborate 82604-100ML
82584-500ML 82604-500ML
for HPCE, 20 mM sodium citrate 82585-100ML for HPCE, 20 mM sodium phosphate 82603-100ML
82585-500ML
Buffer solution HPCE pH 5.0, BioChemika, 82586-50ML for HPCE, 20 mM sodium phosphate 82605-100ML
for HPCE, 20 mM sodium citrate 82586-100ML 82605-500ML
82586-500ML
Buffer solution HPCE pH 6.0, BioChemika, 82588-50ML for HPCE, 20 mM CAPS [3-(cyclohexylamino)-1-propane 82606-100ML
for HPCE, 20 mM sodium citrate 82588-100ML sulfonic acid] 82606-500ML
82588-500ML
Buffer solution HPCE pH 6.5, BioChemika, 82589-50ML for HPCE, 20 mM CAPS [3-(cyclohexylamino)-1-propane 82607-100ML
for HPCE, 20 mM sodium phosphate 82589-100ML sulfonic acid] 82607-500ML
82589-500ML
Buffer solution HPCE pH 7.0, BioChemika, 82591-50ML for HPCE, 20 mM glycine-NaOH 82617-100ML
for HPCE, 20 mM sodium phosphate 82591-100ML
82591-500ML Buffer solution HPCE pH 11.0, BioChemika, 82608-50ML
for HPCE, 20 mM CAPS [3-(cyclohexylamino)-1-propane 82608-100ML
Buffer solution HPCE pH 7.0, BioChemika, 82637-50ML sulfonic acid] 82608-500ML


Detection Kits
Ampliflu Red Western Blot Kit
Classical immunoblotting for protein detection on a membrane is
Marker
50 ng
40 ng
30 ng
20 ng
10 ng
5 ng
1 ng
often based on horseradish peroxidase (HRP) labeled secondary
antibodies. Peroxidase can be detected directly using colorimetric
substrates, or chemiluminescence-based substrates with CCD
camera or X-ray film detection.
The new Ampliflu Red Western Blot Kit contains a peroxidase
Detection Kits
substrate that is enzymatically converted to the highly fluorescent
compound, resorufin (see Figure 1). Fluorescent imaging is
performed on the immunoblot membrane using a laser scanner
FLAG-BAP protein
with an excitation wavelength of 571 nm and an emission filter
of 585 nm (see Figure 2). Other imaging systems with similar
excitation sources and emission filter settings may also be used.
When working with Ampliflu Red, standard protocols for the
immunoblotting-procedure can be used until the detection step.
The membrane is incubated with a mixture of Ampliflu Red and
H2O2 in PBS for 5 minutes. The imaging can then be done via
laser-excitation and an emission filter. No extra time for signal
Figure 2. Detection of FLAG-BAP™ protein (50 ng – 1 ng) by immunoblotting using
accumulation is necessary as it is for chemiluminescent signals. the Ampliflu Red Western blot Kit on Immobilon®-FL PVDF-Membrane. The membrane
The use of Ampliflu Red is suitable for protein quantities between was blocked using 5% BSA in PBS solution and then incubated with monoclonal
1 ng and 1 μg per band of protein. ANTI-FLAG® M2 antibody developed in mouse (Cat. No. F1804, diluted 1:1000).
The secondary antibody used was anti-mouse-IgG-peroxidase developed in rabbit
In order to receive an optimal signal to background ratio, it is (Cat. No. A9044, diluted 1:10,000). Imaging was performed on a FLA-3000 Fuji® laser
scanner with an excitation wavelength of 532 nm and with a 580 nm emission filter.
strongly recommended to use a low-fluorescence PVDF-membrane
for the Western blot transfer and a 5% BSA blocking solution. Ampliflu Red Western Blot Kit
Blocking with 5 % BSA in PBS
Fluorescence
Excitation (1 h-overnight)
Imaging
Short rinse with PBST
Incubate with primary antibody (2-3 h)
Ampliflu Red Resorufin Wash with PBST (3 x 5 min)
Incubate with secondary antibody-HRP (1 h)
sec. antibody–HRP
Wash with PBST (3 x 5 min)
Short rinse with PBS

prim. antibody
5 min incubation with this mixture:

• 1880 µl PBS
• 20 µl Ampliflu Red solution
Protein • 100 µl H2O2 solution
Membrane
Figure 1. Schematic overview for the use of Ampliflu Red. A protein is immobilized by Fluorescent imaging
western blot transfer after SDS-PAGE. The protein is bound with a primary antibody Figure 3. Protocol for the use of Ampliflu Red after SDS-PAGE and Western blot transfer.
and followed by a secondary antibody carrying a horseradish peroxidase (HRP) label. For membranes in the mini-format, the blocking and washing steps were done in a volume
The membrane is incubated in a solution containing Ampliflu Red and hydrogen of 50 mL, whereas, the antibody incubations were done in 25 mL. The Ampliflu Red
peroxide. Ampliflu Red is converted into resorufin by the action of HRP. The resultant incubation mixture in PBS has a total volume of 2 mL. PBST contains 0.1% TWEEN® 20.
fluorescent signal can be detected at the excitation and emission parameters of resorufin
(λex = 571 nm, λem = 585 nm).
Description Cat. No.
Ampliflu Red Western Blot Kit 79454-1KT
TWEEN® 20 P5927-100ML
P5927-500ML
Immobilon®-FL PVDF membrane 05317-10EA

Nitrite/Nitrate Assay Kit
The Nitrite/Nitrate Assay Kit can be used to detect nitrite (NO2-) and Calibration curves
nitrate (NO3-). The detection of NO2- is determined by direct reaction 250
with 2,3-diaminonaphthalene (DAN) (see Figure 1). Fluorescence
of the resulting naphthotriazole can then be measured. The 200
excitation maximum of naphthotriazole is 360-365 nm, with an

150 Nitrite
emission maximum at 410-425 nm. However, in order to reduce
F/u
Nitrate
the fluorescence background of 2,3-diaminonaphthalene and 100
increase detection sensitivity, the use of a 450 nm emission-filter is
recommended. The test is designed for use in 96-well plates, but can 50
Detection Kits
also be done using cuvettes, depending on available equipment.

0
NO3- can only be detected after enzymatic conversion to NO2-. 0 2 4 6 8 10
Subsequent subtraction of NO2- yields the NO3- concentration. µM
The optimal detection range is 1-10 μM for [NO2- + NO3-]. Figure 1. Calibration curves of Nitrite (blue) and Nitrate (red). The graph shows a
linear range for NO2- and NO3- concentrations between 1–10 μM.
- + enzyme - +
NO3 + NADPH + H NO2 + NADP + H2O
-
NO2 + DAN + H
+
Naphthotriazole (fluorescent) + 2 H2O
Nitrite/Nitrate Assay Kit 8
 BioChemika
The assay is designed for the use of clear samples that do not contain any
fluorescent materials, NADPH-consuming enzymes, or other quenching
substances. Cell culture media must be clear and free of phenol red. Any
cell culture medium containing NO3- as a component is not suitable.
for the fluorimetric detection of nitrite and nitrate
The kit contains nitrite solution, nitrate solution, phosphate buffer, nitrate
reductase, FAD, NADPH, diaminonaphthalene, and NaOH.
06239-1KT 1 kit
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Sensor Dyes
Fluorogenic NO Bioimaging Probes
In 1998 R.F. Furchgott, L.J. Ignarro, and F. Murad received the Naphthalenes
Nobel Prize in Physiology and Medicine for their discoveries
2,3-Diaminonaphthalene (DAN) was established as a sensitive
concerning nitric oxide as a signaling molecule in the cardiovascular
probe to measure NO-concentration in vitro and under
system. This indicates the importance the scientific community
physiological conditions.11 The respective 2,3-naphthotriazole (NAT)
has placed on the role of nitric oxide (NO) as a signal transporter
was detected in the low nM concentration range. DAN, however,
in neurons, endothelial cells and in the immune system. NO has
proved to be ill-suited to NO detection in cells due to rapid
Sensor Dyes
been implicated in vasodilation,1 neurotransmission,2 cytotoxicity,
leakage through cell membranes after loading. High background
immune response,3 and inflammation.4-6
fluorescence in biological matrices was also experienced due to its
Within cells, nitric oxide synthase (NOS) catalyzes the conversion rather short excitation and emission wavelengths.
of arginine to citrulline plus NO in the presence of molecular
Therefore, DAN-1 and its cell membrane permeable precursor
oxygen, tetrahydrobiopterin, NADPH, and flavin cofactors.
DAN-1-EE were developed.12 DAN-1 is formed by the hydrolysis
Nε-hydroxy-L-arginine is an intermediate in this reaction. NO has
of DAN-1-EE by cytosolic esterases. DAN-1-T (see Figure 2),
an in vivo half-life of only a few seconds,7 however, since it is
the fluorescent reaction product of DAN-1 with NO, showed
soluble in both aqueous and lipophilic media, it readily diffuses
considerably improved leakage behavior.
through the cytoplasm and across cell membranes. NO has
effects on neuronal transmission as well as synaptic plasticity in 700
the central nervous system. NO activates guanylate cyclase in the 600

fluorescence intensity [a.u.]
vasculature, thus, increasing the synthesis of cGMP, which in turn

500
induces smooth muscle relaxation and vasodilation. NO toxicity is
related to its ability to combine with superoxide anions to form 400
peroxynitrite, an oxidizing free radical that can damage DNA and 300
other cellular constituents.
200
The NO molecule is unstable and occurs in very low concentrations
100
in biological systems. In order to detect it in real time, methods
with high sensitivities and specificities are required. Fluorescence 0
0 10 20 30 40 50 60 70
microscopy seems to be the method of choice, provided suitable
[min]
NO probes are available. This high spatial-temporal resolution
Figure 2. Time course of formation of fluorescent DAN-1-T from DAN-1 upon addition
technique allows for reliable imaging of in situ NO concentrations. of final concentration of 5 mM (green line) or 50 mM (red line) respectively of a NO
forming agent (NONOate)[12].
Research into the design of sensitive NO probes revealed a
reaction involving vicinal aromatic diamino compounds8,9
that allow for selective trapping and detection of NO.
Figure 1 illustrates this reaction using the novel NO probe
5,6-diaminofluorescein diacetate (DAF-2-DA) as an example.
NH2 NH2 N NH
H2N H2N N
intracellular
esterases NO / O2
O O O
O O O
AcO O OAc HO O OH HO O OH
DAF-2-DA DAF-2 DAF-2 T
Figure 1. Principle of intracellular NO measurement, shown with the probe DAF-2 DA[10].
Lipophilic, non-fluorescent DAF-2-DA permeates the cell membrane and is then hydrolyzed
by cytosolic esterases to the weakly fluorescent probe DAF-2. The presence of NO radicals
transforms it to the strongly fluorescent triazole DAF-2 T.

Xanthenes 1000
900
Fluorescein and rhodamine-based probes have been developed
fluorescence intensity (a.u.)

800
in order to eliminate some drawbacks of naphthalene-based NO
700
probes, such as cytotoxicity, strong autofluorescence, a small
600
extinction coefficient, poor solubility in neutral buffer and short
500
excitation/emisson wavelengths.10,13-16
400
Limit of detection (LOD) of NO by DAF-2 is 5 nM in vitro, in the 300
absence of absorbing side products. Oxidized forms of NO such 200
as NO2- and NO3-, as well as reactive oxygen species such as O2·-, 100
H2O2, and ONOO- do not react with DAF-2 to give a fluorescent 0
Sensor Dyes
2 3 4 5 6 7 8 9 10 11
product. Therefore, under physiological conditions fluorescent
pH
DAF-2 T is only formed in the presence of NO.
Figure 3. pH dependence of fluorescence intensity (a.u.) of DAF-2 (green), DAR-1
A fluorescent response was detected when cultured, smooth (orange) and DAR-2 (pink)[11].
aortic rat muscle cells loaded with DAF-2-DA were viewed
using a confocal laser scanning microscope in the presence of References
endotoxins and cytokines. The addition of L-arginine resulted in a 1. Palmer, R.M., et al., Nature, 327, 524 (1987).
2. J. Garthwaite, J., et al., Nature, 336, 385 (1988).
sharp increase of the fluorescence signal, whereas supplementary
3. Marletta, M.A., et al., Biochemistry, 27, 8706 (1988).
addition of N-monomethyl-L-arginine (L-NMMA) resulted in no 4. Stuehr, D.J., Ann. Rev. Pharmacol. Toxicol., 37, 339 (1997).
5. Marletta, M.A., et al., Curr. Opin. Chem. Biol., 2, 656 (1998).
signal increase, clearly demonstating the inhibition of NO-synthase. 6. Geller, D.A. and Billiar, T.R., Cancer Metastasis Rev., 17, 7 (1998).
7. Mordvintcev, P., et al., Anal. Biochem., 199, 142 (1991).
The fluorescein type probe (DAF-2) shows a bright fluorescence 8. Misko, T.P., et al., Anal. Biochem., 214, 11 (1993).
signal; while the rhodamine type probes (DAR-1 and DAR-2) excel 9. Miles, A.M., et al., Methods Enzymol., 268, 105 (1996).
10. Kojima, H., et al., Anal. Chem., 70, 2446 (1998).
with high photostability and their applicability over a broad pH 11. Andrew. P.J., et al., FEBS Lett., 408, 319 (1997).
12. Kojima, H., et al., Biol. Pharm. Bull., 20, 1229 (1997).
range (see Table 1, Table 2 and Figure 3). 13. Kojima, H., et al., Tetrahedron Lett,. 41, 69 (2000).
14. Kojima, H., et al., Anal. Chem., 73, 1967 (2001).
λabs., max. [nm] ε [x104 M-1cm-1] λem., max. [nm] rel. quantum yields 15. Kojima, H., et al., Chem. Pharm. Bull., 46, 373 (1998).
probe 16. Nakatsubo, N., et al., FEBS Lett., 427, 263 (1998).
diamine triazole diamine triazole triazole diamine triazole
DAF-2 486 491 7.9 7.3 513 0.005 0.92
DAR-1 550 556 12 8.7 575 0.004 0.25 Fluorogenic Probes
DAR-2 549 552 10 7.1 571 0.006 0.34
Name Cat. No.
Table 1. Spectral properties of DAF and DAR probesa)b)
DAR-1 09014-1MG-F
a) Data from reference.11 DAR-2 08715-1MG-F
b) D
ata at 20 °C in 0.1 M phosphate buffer, pH 7.4; quantum yields derived by 08715-5MG-F
comparison with known quantum yield of rhodamin B in ethanol.
4,5-Diaminofluorescein D224-1MG
5,6-Diaminofluorescein diacetate 50277-1MG-F

DAF’s DAR’s
λex [nm] ~490 ~550 4,5-Diaminofluorescein diacetate D2813-1MG
λem [nm] ~515 (green) ~575 (red)
2,3-Diaminonaphthalene 88461-25MG-F
suitable pH range > pH 6 > pH 4 88461-100MG-F
detection limit for NO ~5 nM (DAF-2, in vitro) ~10 nM (DAR-M, in vitro)
Ethyl 4-[(3-amino-2-naphthyl)aminomethyl]benzoate 16513-1MG-F
photostabilityb) photobleached more than 70% not photobleached
hydrochloride 16513-10MG-F
distribution nucleus/cytosol equal cytosol > nucleus
4-[(1-Naphtho[2,3-d]triazol-1-yl)methyl]benzoic acid 36495-1MG-F
Table 2. General Properties of DAF and DAR probesa)b) 36495-10MG-F
a) Data from references.10-12

b) Based on fluorescence intensity measurements after exposure to sunlight for 3 h.

Additional Reagents for NO Bioimaging
Name Cat. No. Name Cat. No.
3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene A5581-10MG S-Nitroso-N-acetyl-DL-penicillamine N3398-25MG

A5581-50MG N3398-50MG
3-Bromo-7-nitroindazole B2050-10MG NOC-5 A5456-10MG
Cyclosporin A 30024-25MG Sodium nitroprusside dihydrate 71778-25G

30024-100MG 71778-100G
Diethylamine NONOate diethylammonium salt D5431-50MG Spermidine trihydrochloride 85580-5G

85580-25G
Diethylamine NONOate sodium salt hydrate D184-25MG
D184-50MG Spermine S3256-1G
S3256-5G
3-Ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene E3145-10MG S3256-25G
Sensor Dyes
(±)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide E2895-10MG Spermine tetrahydrochloride S2876-1G
S2876-5G
(±)-(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl- E3020-10MG
S2876-10G
nicotinamide
S2876-25G
Ketorolac tris salt K1136-1G S2876-100G
K1136-5G
Spermine–Nitric oxide complex hydrate S150-25MG
MAHMA NONOate M1555-50MG
Streptozocin S0130-50MG
3-Morpholinosydnonimine hydrochloride M5793-25MG S0130-100MG
M5793-100MG S0130-500MG
S0130-1G
Nitric Oxide Synthase Detection Kit, Isotopic NOS1-1KT S0130-5G
Sulfo NONOate disodium salt S8432-50MG
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Chelators and Ion Probes
Ions play a key role in important biological processes and in the investigation of biochemical structures and activities. Calcium ions, in
particular, are of extraordinary importance in many key cellular processes. Since the detection of calcium concentration by fluorescence
can be accomplished using various techniques, such as microscopy, flow cytometry and spectroscopy, fluorescent indicators can function in
widespread applications for studying biological processes. Sigma® Life Science offers a comprehensive list of products for a broad range of
ion sensitive applications.
BAPTA-AM - A1076-25MG
1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid - 40843-10MG-F

Sensor Dyes
tetrakis(acetoxymethyl) ester
1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid - 16609-10MG-F

tetrakis(acetoxymethyl) ester
1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid - A4926-250MG

A4926-1G
A4926-5G
1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrasodium salt - 14513-250MG

14513-1G
4,4′′-Bis(4,4,5,5,6,6,6-heptafluoro-1,3-dioxohexyl)-o-terphenyl-4′-sulfonyl chloride 386 / 613 nm in 0.1 M Tris pH 8.0, Eu3+ 59752-5MG-F
N,N′-Bis(salicylidene)ethylenediamine - 236071-10G
Calcein - C0875-5G
C0875-10G
C0875-25G
Calcein disodium salt - 21030-1G-F

21030-5G-F
21030-100G-F
Calcein-AM 496 / 516 nm in 0.1 M Tris pH 8.0, esterase; Ca2+ 17783-1MG

17783-5MG
Calcein AM solution - C1359-100UL
Calcein Blue - M1255-1G

M1255-10G
2,3-Diaminonaphthalene 364 / 406 nm in 0.5 M Na carbonate pH 10.0 (after derivatization 88461-25MG-F

with NaNO2 in 0.1 M HCl) 88461-100MG-F
1-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide 350 / 451 nm in 0.1 M borate pH 8.0 (quenching with Cl-) 46123-100MG-F
Fluo-3 506 / 526 nm in 0.1 M Tris pH 8.0; esterase 30 mM Ca2+ 46393-1MG-F

46393-5MG-F
Fluo 3-AM 506 / 526 nm in 0.1 M Tris pH 8.0; esterase 30 mM Ca2+ 46394-1MG
Fura 2 pentapotassium salt 355 / 495 nm in 0.1 M Tris pH 8.0; Ca2+ 17195-1MG
Fura 2-AM 355 / 495 nm in 0.1 M Tris pH 8.0; esterase; 10 mM Ca2+ 47989-1MG-F
Indo 1-AM - I3261-250UG

I3261-1MG
7-(Isobutoxycarbonyloxy)-3H-phenoxazin-3-one 500 / 593 nm in 0.1 M Tris pH 8.0 (after cleavage by esterase) 79972-1MG-F
8-Methoxypyrene-1,3,6-trisulfonic acid trisodium salt 404 / 431 nm in H2O 65325-100MG-F
Nitr 5/AM - 72543-1MG

72543-5MG
PBFI-AM 370 / 540 nm in methanol 76275-1MG
29H,31H-Phthalocyanine - 253103-1G
253103-5G
1,3,6,8-Pyrenetetrasulfonic acid tetrasodium salt hydrate - 82658-1G
2,6-Pyridinedicarboxylic acid - P63808-25G

P63808-100G
P63808-500G
Quin-2 332 / 493 nm in 0.1 M Tris pH 8.0, 10 mM Ca2+ 08520-50MG
Quin 2-AM - Q4875-10MG
Q4875-50MG
SBFI-AM - S1148-.1MG
S1148-1MG
5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) - 323497-100MG

323497-250MG
4,4,4-Trifluoro-1-(2-naphthyl)-1,3-butanedione - 343633-5G
343633-25G

pH Indicators
pH indicators show a change in their characteristic optical spectra depending on pH. Fluorescent pH indicators allow a more sensitive
detection compared to chromogen pH indicators which are often called for in cellular studies. Intercellular pH changes play an important
role in apoptosis and ion transport mechanisms and are a characteristic of many diseases.
9-Aminoacridine hydrochloride hydrate - A38401-5G

A38401-25G
8-Anilino-1-naphthalenesulfonic acid ammonium salt - 10417-5G-F

10417-25G-F
10417-100G-F
Sensor Dyes
8-Anilino-1-naphthalenesulfonic acid hemimagnesium salt hydrate - A5144-5G
A5144-25G
2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein - C3411-1MG
2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester 482 / 528 nm in 0.1 M Tris pH 8.0 (esterase) 14562-1MG
2′,7′-Bis(3-carboxypropyl)-5(6)-carboxyfluorescein 480 / 527 nm in 0.1 M Tris pH 9.0 17283-5MG

17283-25MG
5(6)-Carboxy-2′,7′-dichlorofluorescein 504 / 529 nm in 0.1 M Tris pH 8.0 21882-100MG-F
5(6)-Carboxyfluorescein 492 / 517 nm in 0.1 M Tris pH 8.0 21877-1G-F

21877-5G-F
5-Carboxyfluorescein diacetate - C4916-25MG

C4916-100MG
5(6)-Carboxyfluorescein diacetate 492 / 517 nm in 0.1 M Tris pH 8.0 (esterase) 21879-25MG-F

21879-100MG-F
6-Carboxyfluorescein diacetate - C5041-25MG

C5041-100MG
5(6)-Carboxyfluorescein N-hydroxysuccinimide ester 492 / 517 nm in DMF 21878-25MG-F

21878-100MG-F
5(6)-Carboxynaphthofluorescein 598 / 668 nm (basic) 21932-25MG-F

512 / 567 nm (acid/neutral) 21932-100MG-F
Chromoionophore I - 27086-10MG-F
27086-100MG-F
3-Cyanoumbelliferone 408 / 450 nm in methanol 28605-100MG

28605-500MG
3,6-Diacetoxyphthalonitrile - D0679-5MG
6,8-Dihydroxy-1,3-pyrenedisulfonic acid disodium salt 458 / 498 nm in 0.1 M Tris pH 8.0 37920-100MG
405 / 456 nm in 0.1 M citrate pH 3.0 37920-500MG
N,N-Dimethyl-6-propionyl-2-naphthylamine 361 / 498 nm in methanol 41525-25MG

41525-100MG
6-Dodecanoyl-N,N-dimethyl-2-naphthylamine 366 / 497 nm in methanol 40227-100MG
Eosin diacetate - 45244-1G

45244-5G
Fluorescein diacetate - F7378-5G

F7378-10G
F7378-25G
F7378-100G
7-Hydroxycoumarin-3-carboxylic acid 386 / 448 nm in 0.1 M Tris pH 9.0 55155-100MG-F
7-Hydroxycoumarin-3-carboxylic acid N-succinimidyl ester 386 / 448 nm in 0.1 M Tris pH 9.0 55156-25MG-F
55156-100MG-F
7-Hydroxy-4-methyl-3-coumarinylacetic acid 360 / 450 nm in 0.1 M Tris pH 9.0 55634-100MG
7-Hydroxy-4-methyl-2(1H)-quinolone 351 / 428 nm in 0.1 M Tris pH 9.0 55627-100MG

55627-500MG

pH Indicators, continued
7-Hydroxy-N-octadecylcoumarin-3-carboxamide 359 / 406 nm in chloroform/DMSO (1:1) 56059-100MG-F

56059-500MG-F
8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt - H1529-1G
Naphthofluorescein 594 / 663 nm in 0.1 M Tris pH 9.0 70420-100MG

70420-500MG
8-Octadecyloxypyrene-1,3,6-trisulfonic acid trisodium salt 404 / 434 nm in H2O 74758-50MG
2-Phenylphenol - P28263-500G
P28263-2KG
Sensor Dyes
Rhodamine B octadecyl ester perchlorate 554 / 575 nm in methanol 83685-10MG
Tetramethylrhodamine ethyl ester perchlorate 540 / 595 nm in DMSO 87917-25MG
Tetramethylrhodamine methyl ester perchlorate - T5428-25MG
6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt - T9792-250MG

T9792-1G
Liphophilic and Membrane Probes

Fluorescent dyes are frequently used for the study of plasma membranes, intracellular membranes or artificial membranes. They allow the
structural and biophysical investigation of signal transfer within membranes as well as the monitoring of lipid-based metabolism. Sigma®
Life Science offers a comprehensive range of membrane dyes, including a large series of potential-sensitive fluorophores, designed for the
determination of transmembrane potential in living cells.
4-(2-Aminoethylamino)-7-(N,N-dimethylsulfamoyl)benzofurazan 406 / 581 nm in 0.1 M phosphate pH 7.0 93088-25MG-F
6-Aminofluorescein 490 / 520 nm in 0.1 M Tris pH 9.0 07985-1G

07985-5G
7-Amino-4-methylcoumarin 365 / 440 nm in ethanol A9891-250MG

A9891-500MG
A9891-1G
A9891-5G
7-Amino-4-methyl-3-coumarinylacetic acid 350 / 433 nm in methanol 08445-100MG

08445-500MG
8-Aminonaphthalene-1,3,6-trisulfonic acid disodium salt 356 / 512 nm in 0.1 M phosphate pH 7.0 08658-500MG
(S)-(+)-4-(3-Amino-pyrrolidino)-7-nitrobenzofurazan 490 / 535 nm in acetonitrile 76488-10MG-F
9-Anthracenecarboxaldehyde - 278688-5G
278688-25G
278688-100G
2-Anthracenesulfonyl chloride 382 / 421 nm in acetonitrile (after derivatization with hexylamine) 06479-100MG-F
4-Bromomethyl-7-methoxycoumarin 322 / 395 nm (after derivatization) 235202-250MG

235202-1G
235202-5G
5(6)-Carboxy-X-rhodamine 570 / 595 nm in 0.1 M Tris pH 8.0 21965-25MG-F

21965-100MG-F
5(6)-Carboxytetramethylrhodamine 543 / 570 nm in methanol 21953-100MG-F

21953-500MG-F
Chromeon 641-lipophil 641 / 659 nm in chloroform 67589-1MG-F
Chromeon 783-lipophil 783 / 807 nm in methanol 80108-1MG-F
Coumarin-6-sulfonyl chloride 360 / 405 nm in acetonitrile (after derivatization with hexylamine) 66408-10MG-F
5-([4,6-Dichlorotriazin-2-yl]amino)fluorescein hydrochloride - D0531-100MG

D0531-500MG
D0531-1G
7-(Diethylamino)coumarin-3-carboxylic acid 409 / 473 nm in 0.1 M Tris pH 9.0 36799-100MG-F
7-(Diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester 445 / 482 nm in 0.1 M phosphate pH 7.0 36801-25MG
36801-100MG
3,3′-Diethylthiacarbocyanine iodide 554 / 577 nm in phosphate buffer/SDS pH 7.0 36809-250MG
3,3′-Diethylthiadicarbocyanine iodide - 173754-1G
3,3′-Dihexyloxacarbocyanine iodide - 318426-250MG

318426-1G

N-(7-Dimethylamino-4-methyl-3-coumarinyl)maleimide 398 / 482 nm in 0.1 M phosphate pH 7.0 (after derivatization with 39145-10MG
2-mercaptoethanol)
N,N′-Dimethyl-9,9′-biacridinium dinitrate - M8010-500MG

M8010-1G
M8010-5G
3,3′-Dioctadecyloxacarbocyanine perchlorate - D4292-20MG
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate 550 / 565 nm in phosphate buffer/SDS pH 7.0 42364-100MG
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, pyrene-labeled 323 / 378 nm in methanol 72674-1MG-F
1,6-Diphenyl-1,3,5-hexatriene - D208000-1G
D208000-5G
43095-1G
Sensor Dyes
Diphenylmaleic anhydride 284 / 506 nm (after derivatization with hexylamine)
43095-5G
3,3′-Dipropylthiacarbocyanine iodide - 318434-250MG
3,3′-Dipropylthiadicarbocyanine iodide 500 / 705 nm in DMF 43608-100MG
6-Dodecanoyl-N,N-dimethyl-2-naphthylamine 366 / 497 nm in methanol 40227-100MG
Eosin 5-isothiocyanate - 45245-50MG
Fluorescein isothiocyanate isomer I - F7250-50MG

F7250-100MG
F7250-250MG
F7250-500MG
F7250-1G
F7250-5G
N-(Iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid 337 / 498 nm (after derivatization with 2-mercaptoethanol) 57672-1G-F
JC-1 - T4069-5MG
5-Maleimido-eosin 524 / 545 nm (after derivatization with 2-mercaptoethanol) 63184-10MG

63184-25MG
Merocyanine 540 - 323756-100MG

323756-250MG
7-Methoxycoumarin-3-carboxylic acid 330 / 402 nm in 0.1 M Tris pH 9.0 64948-100MG

64948-500MG
10-Methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate - 68617-25MG

68617-100MG
1-Methylpyrene 346 / 378 nm in DMSO 69025-100MG
Mito Red 569 / 594 nm in DMSO 53271-8X50UG-F
3-Morpholinobenzanthrone 439 / 632 nm in dimethyl sulfide 50726-1MG-F

50726-5MG-F
6-(7-Nitrobenzofurazan-4-ylamino)hexanoic acid 466 / 535 nm in methanol 72964-100MG

72964-500MG
1-Pyrenebutyric acid - 257354-1G

257354-5G
1-Pyrenedecanoic acid 340 / 376 nm in methanol 82660-25MG
1-Pyrenedodecanoic acid 339 / 377 nm in methanol 82663-25MG
1-Pyrenesulfonic acid sodium salt 314 / 376 nm in methanol 82657-1G
N-(1-Pyrenyl)maleimide - P7908-100MG
P7908-500MG
P7908-1G
Rhodamine 123 - 83702-10MG

83702-50MG
Rhodamine B isothiocyanate - R1755-100MG

R1755-500MG
R1755-1G
Rhodamine B octadecyl ester perchlorate 554 / 575 nm in methanol 83685-10MG
Tetramethylrhodamine ethyl ester perchlorate 540 / 595 nm in DMSO 87917-25MG
Tetramethylrhodamine isothiocyanate mixed isomers 529 / 596 nm in DMSO 87918-10MG

87918-50MG
Tetramethylrhodamine methyl ester perchlorate - T5428-25MG
6-(p-Toluidino)-2-naphthalenesulfonyl chloride 381 / in methanol 04076-50MG

04076-250MG
N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenylammonium 355 / 430 nm in methanol 43060-25MG-F

p-toluenesulfonate

New Product Corner
Atto 488 Protein Labeling Kit 8 CYPMPO 8
This kit contains sufficient amounts of reactive dye, buffers and protein 5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline
purification sets for performing five labeling reactions (1mg protein each) N-oxide; 2-(5,5-Dimethyl-2-oxo-2λ5-[1,3,2]dioxaphosphinan-2-yl)-2-
and for the subsequent purification of the labeled protein. methyl-3,4-dihydro-2H-pyrrole 1-oxide
38371-1KT 1 kit [934182‑09‑9] C10H18NO4P FW 247.23
Atto 550 Protein Labeling Kit 8

 ≥97% (HPLC)
CYPMPO is designed for radical detection. It is a free radical spin trap with
This kit contains sufficient amounts of reactive dye, buffers and protein excellent trapping capabilites toward hydroxyl and superoxide radicals in
purification sets for performing five labeling reactions (1 mg protein each) biological and chemical systems.
01872-1MG 1 mg
New Product Corner
51146-1KT 1 kit
Atto 633
This kit contains sufficient amounts of reactive dye, buffers and protein  BioChemika, for fluorescence
purification sets for performing five labeling reactions (1 mg protein each) 18620-1MG 1 mg
N-Biotinyl-3,6,9-trioxaundecane-1,11-
68616-1KT 1 kit
diamine trifluoroacetate salt solution
Atto 647N Protein Labeling Kit 8 N-{2-[2-(2-(2-Aminoethoxy)ethoxy)ethoxy]ethyl}biotinamide
This kit contains sufficient amounts of reactive dye, buffers and protein trifluoroacetate salt
purification sets for performing five labeling reactions (1 mg protein each) [945462‑84‑0] C18H34N4O5S · C2HF3O2 FW 532.57
 ≥95.0% (TLC), 25 mg/mL in DMSO
76508-1KT 1 kit Nucleophilic biotinylation reagent for the attachment of cell surface
glycosides to ELISA-type surfaces via a hydrophilic spacer/linker.
38801-25MG-F 25 mg
This kit contains sufficient amounts of reactive dye, buffers and protein
38801-100MG-F 100 mg
purification sets for performing five labeling reactions (1 mg protein each)
and for the subsequent purification of the labeled protein. N-Biotinyl-ethylenediamine trifluoroacetate salt
51253-1KT 1 kit N-(2-Aminoethyl)biotinamide trifluoroacetate salt
C12H22N4O2S · C2HF3O2 FW 400.42
This kit contains sufficient amounts of reactive dye, buffers, and protein  ≥96.5% (HPLC)
purification sets for performing five labeling reactions (1 mg protein each) Nucleophilic biotinylation reagent for the conjugation of various substrates and
and for the subsequent purification of the labeled protein. their identification and quantification using biotin-binding fluorescent labels
73919-1KT 1 kit 08599-100MG 100 mg
08599-500MG 500 mg
Rhodol 8
3′-Amino-6′-hydroxy-fluoran Nε-(N-(+)-Biotinyl-6-aminohexanoyl)-Nα,Nα-
[3086‑44‑0] C20H13NO4 FW 331.32 bis(carboxymethyl)-L-lysine tripotassium salt
Biotin-X-NTA
 BioChemika, ≥85% (HPCE)
[856661‑92‑2] C26H40K3N5O9S FW 715.98
Rhodol is a strongly fluorescent dye. It is a structural hybrid of fluorescein
and rhodamine.  BioChemika, ≥98.0% (TLC)
67806-5MG 5 mg Hetero-bifunctional linker for His-tagged proteins on one side and (strept)
avidin conjugates
N-(5-Fluoresceinyl)maleimide 8 51410-5MG 5 mg
5-Maleimido-fluorescein 51410-25MG 25 mg
[75350‑46‑8] C24H13NO7 FW 427.36
Atto 655 Phalloidin 8
 ≥90% (HPLC)
corresponds for coupling to thiols  BioChemika, ≥95.0% (HPCE, sum of isomers)
38132-25MG 25 mg Fluorescent labeled phalloidin is used for the specific labeling of microtubuli.
18846-10NMOL 10 nmol
N-(4-Methylumbelliferyl)maleimide
7-Maleimido-4-methylcoumarin Ovalbumin-Atto 488 conjugate 8
[211565‑47‑8] C14H9NO4 FW 255.23 Fluorescent labeled ovalbumin is used as endocytic tracer.
corresponds for coupling to thiols free of unconjugated dye
92333-25MG 25 mg 41235-2MG 2 mg
Ovalbumin-Atto 594 conjugate 8
Fluorescent labled ovalbumin is used as endocytic tracer.

free of unconjugated dye
76503-2MG 2 mg

Books
Green Fluorescent Protein: Properties, Applications Molecular Fluorescence: Principles and Applications
and Protocols, 2nd ed. Fluorescence spectroscopy is an important tool of investigation in many
This text tackles both theory and practice, offering numerous case studies, areas. Its advantage is extremely high sensitivity and selectivity - even
examples, illustrations, and troubleshooting tips. It examines how Ps are single molecules can be detected - and it achieves a high spatial resolution
tailored for specific systems and used to maximize expression, and how and time resolution in combination with microscopic techniques or
variants are generated with altered properties. The text also explores laser techniques, respectively. In biochemistry and molecular genetics,
new ways to use FPs and methods for enhancing detection in a variety fluorescence spectroscopy has become a dominating technique. Together
of organisms and cell types. This updated and expanded edition places with the latest imaging techniques, fluorescence spectroscopy allows a
emphasis on the rise of FPs and their applications in industry and biosensor real-time observation of the dynamics of intact biological systems with an
research. It provides new chapters on FPs, biosensors, and advances in the unprecedented resolution.
use of FPs in the biotechnology and pharmaceutical industries, detailed
Books
information about protocols using FPs, and three-dimensional structure, and
molecular biology and mutation of FPs.
M0566-1EA 1 ea
Principles of Fluorescence Spectroscopy 3rd ed.

Z705217-1EA 1 ea The third edition of the established classic text reference, will enhance
upon the earlier editions’ successes. It will maintain the emphasis on basics,
Luminescence Biotechnology: Instruments and Applications
while updating the examples to include recent results from the literature.
The dangers inherent in radioactivity-based methods have caused a shift This edition also includes new chapters on single molecule detection,
towards luminescence measurements and visualization techniques. This fluorescence correlation spectroscopy, novel probes and radiative decay
up-to-date review covers the fundamentals of different luminescence- engineering. The full color text features the following: - Problem sets
based assay systems, calculation methods, and instruments through the following every chapter - Glossaries of commonly used acronyms and
spectrum of applications and research advances. Topics include gene and mathematical symbols - Appendices containing a list of recommended
protein assays, oxidative stress and tissue aging, applications of luminescent books which expand on various specialized topics - Sections describing
microspheres, and proton image analysis. This book clearly identifies the advanced topics will indicate as such, to allow these sections to be skipped
advantages of luminescence over other assay techniques, discusses its in an introductory course, allowing the text to be used for classes of
potential pitfalls, and illustrates the broad range of its utility. different levels.
L3165-1EA 1 ea Z705969-1EA 1 ea
Protein Localization by Fluorescence Microscopy:

A Practical Approach
The ability to determine the location of gene products within a cell by the
use of antibodies or by production of chimeras with green fluorescent
protein is a vital step towards understanding what they do. This guide
provides detailed, practical advice from choosing the right equipment to
interpreting results. It balances the advantages of a range of techniques,
including live cell work, against potential pitfalls, offering invaluable “tricks
of the trade” along the way.
Z340847-1EA 1 ea

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Life Science

Innovative Solutions for Fluorescent and

Your gateway to Biochemicals and Reagents

New NIR Fluorescence Dyes 6

Detection Web Resource Detection Kits 17

Our Innovation, Your Research — Shaping the Future of Life Science 3

(see Figure 1). Nancy-520 is provided as a 5000× stock solution 4361 –

Nancy-520 + dsDNA in TBE, pH 8.3

Nancy-520 can be used in a standard post-electrophoresis staining 4500

electrophoresis run without any further steps. Nancy-520 allows 500

more detection possibilities than SYBR® Green. Detection is 0

4 sigma.com/lifescience Order: sigma.com/order Technical service: sigma.com/techinfo

GenElute™ Agarose Spin Columns 56500-70EA

Gel Loading Buffer G2526-5ML

Lambda DNA Hind III Digest D9780-20UG

Tris-Borate-EDTA buffer T7527-1L

Tris Acetate-EDTA buffer T8280-100ML

DNA and RNA Intercalating Fluorophores

Description λex/λem (nm) Cat. No.

Acridine Orange hemi(zinc chloride) salt - 158550-10G

Atto-Dino 2 solution - 83399-200UL-F

Atto-Dino 4 solution - 83392-200UL-F

bisBenzimide H 33258 355 / 465 nm in TE buffer; DNA 14530-100MG

bisBenzimide H 33342 trihydrochloride 355 / 465 nm in TE buffer; DNA 14533-100MG

4′,6-Diamidino-2-phenylindole dihydrochloride 374 / 461 nm in 10 mM Tris; 1 mM EDTA; pH 8.0; DNA 32670-5MG-F

Dimidium bromide 306 / 612 nm in 50 mM Tris pH 8.0 (DNA) 41785-250MG

Ethidium bromide 530 / 600 nm in 50 mM phosphate buffer pH 7.0 46065-1G

Ethidium bromide solution 530 / 600 nm in 50 mM phosphate buffer pH 7.0 46067-50ML-F

Ethidium homodimer 528 / 598 nm in TE buffer; DNA 46043-1MG-F

Propidium iodide - P4170-10MG

Propidium iodide solution - P4864-10ML

Pyrene 338 / 375 nm in DMSO 82648-1G

SYBR® Green II RNA gel stain - S9305-.5ML

Our Innovation, Your Research — Shaping the Future of Life Science 5

fluorescence intensity [au]

Investigation of optimal dye/protein (D/P) ratio of antibody

Table 1. Spectroscopic data of Tracy fluorophores, useful for dye/protein ratio

Several major advantages of the red emitting fluorescent dyes over

6 sigma.com/lifescience Order: sigma.com/order Technical service: sigma.com/techinfo

New NIR Fluorescence Dyes

Life Science Innovations and BioFiles

Our Innovation, Your Research — Shaping the Future of Life Science 7

Nα, Nα-bis(carboxy­methyl)-L-lysine Nickel(II) com­plex - Tracy 652 conjugate

Tracy 652 (free acid) 8 Tracy Protein Labeling Kits

Detection Web Resource

Over 1,000 Products for Find the Optimal Probe or Label!

To learn more, visit sigma.com/fluorescence

8 sigma.com/lifescience Order: sigma.com/order Technical service: sigma.com/techinfo

many detection applications, including carbohydrate, histochemical

Description λex/λem (nm) Cat. No.

Concanavalin A-Atto 488 conjugate 501 / 523 nm in PBS 40634-1MG

Concanavalin A-Atto 565 conjugate 563 / 592 nm in PBS 69535-1MG

+7 (495) 621 5828

■ Glycan Labeling and Analysis

■ Glycoprotein Purification and

Tools for Glycoproteomics and Glycomics

Tel: (+81) 3 5796 7300

spectroscopy, and other key techniques

■ Glycoprotein Purification and

■ Chemical and Enzymatic

Visit sigma.com/glycomanual and request your copy.

Our Innovation, Your Research — Shaping the Future of Life Science

10 sigma.com/lifescience Order: sigma.com/order Technical service: sigma.com/techinfo

Nα, Nα-bis(carboxymethyl)-L-lysine Nickel(II) complex - Tracy 652 conjugate

L UCY 569 is particularly well suited for protein quantitation, displaying a

Inadequate purity for application as a standard