innovative solutions for fluorescent and chemiluminescent applications - biofiles issue 4.1

Green fluorescent protein (GFP) in a Pacific jellyfish, Aequorea victoria

Volume 4, Number 1

BioFilesLife Science

Innovative Solutions for Fluorescent and Chemiluminescent Applications

DNA Detection

New NIR Fluorescence Dyes

Carbohydrate Detection

Protein Detection

Isoelectric Focusing

Detection Kits

Sensor Dyes

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2

Life Science

BioFilesVolume 4, Number 1

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

New Fluorescently Labeled Lectins 10

Fluorescent Labels for Carbohydrates 11

Protein Detection 12

LUCY® Protein Stains 12

Isoelectric Focusing 14

Fluorescent IEF-Markers 15

Buffers for Capillary Zone Electrophoresis 16

Detection Kits 17

Ampliflu Red Western Blot Kit 17

Nitrite/Nitrate Assay Kit 18

Sensor Dyes 19

Fluorogenic NO Bioimaging Probes 19

Chelators and Ion Probes 22

pH Indicators 23

Liphophilic and Membrane Probes 24

New Product Corner 26

Books 27

Authors of articles: Alex Rueck, Ph.D.; Monika Baeumle, Ph.D.; Bernhard Schoenenberger, Ph.D.

Other technical contribution:Klaus Trummler, Ph.D.; Jakob Zbären, Inselspital Bern, Switzerland

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Detection Web Resource

The detection web resource includes selection tables and learning center pages for a large number of luminescent probes and indicators, stains and dyes, microparticles, embedding media, and substrates.

The detection resource also contains two useful tools for designing and implementing a detection technique that is right for your research.

Method-to-Product Guide

Enables users to select the correct product based on the method of detection being used or vice versa.

For example, the method-to-product guide can help select the correct stains and labels for detecting proteins by flow cytometry or the correct probes for lipophilic and membrane detection by microscopy.

Wavelength Index

Quickly find excitation and emission wavelengths for a large number of detection products.

Detection products and web tools can be found at sigma.com/fluorescence

no

rm. f

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wavelength (nm)550 600 700 750650

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

Intro

du

ction

The 2008 Nobel Prize for Chemistry was awarded to Osamu Shimomura, Martin Chalfie and Roger Tsien for the discovery and development of the green fluorescent protein, GFP. GFP was first isolated from the jellyfish, Aequorea victoria in 1962 by

Shimomura. Since then, it has become one of the most important tools used in life science research. GFP enables researchers to investigate processes within living cells. This award emphasizes the importance of luminescent techniques as one of the most important, widespread and fast-growing analytical tools in life science and analytical biochemistry.

Fundamental requirements in fluorescent techniques are high sensitivity and selectivity, allowing a scientist to track molecular interactions, mobility, and conformational changes of 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, Sigma-Aldrich® has been a leading supplier of fluorescent probes for many years. In order to ensure the highest level of performance, we continuously investigate and develop new products and optimize application protocols for the fields of detection and biochemistry. Our Detection Center of Excellence located in Buchs, Switzerland has risen to this challenge and has developed a series of novel fluorescent dyes.

IntroductionMonika Baeumle, Ph.D.Product Manager, [email protected]

Single molecule studies are one

of the hottest topics in biological

sciences because they bring us closer

to understanding cellular processes.

Read and comment on this exciting

application of detection research at

the BioBlog.

This issue of BioFiles focuses on these new products for fluorescent bioanalytical techniques

� Nancy-520 for DNA staining

� Fluorescent near-IR dyes Tracy 645 and Tracy 652

� Atto-dye conjugated lectins for carbohydrate analysis

� LUCY® dyes for enhanced protein staining

� Fluorescent isoelectric focusing (IEF) markers

� Ampliflu Red Western Blot Kit

� Highly sensitive Fluorogenic NO Bioimaging Probes for intracellular measurements

We hope that you will find our articles and products to be interesting and helpful. For further information, please visit our detection web resource at sigma.com/fluorescence.

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

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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 maximum at 520 nm and an emission maximum at 560 nm (see Figure 1). Nancy-520 is provided as a 5000× stock solution (500 μl), which is sufficient for 50 agarose gels. The limit of detection is 0.5 ng/band of dsDNA. In addition, Nancy-520 can be used to determine dsDNA concentrations in solution.

Nancy-520 + dsDNA in TBE, pH 8.3

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0.2

0.4

0.6

0.8

1

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400 450 500 550 600 650 700

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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 fluorescence spectrophotometer with 2 mL of TBE, pH 8.3. The excitation spectrum was measured at a fixed emission wavelength of 560 nm, and the emission spectrum was measured at a fixed excitation wavelength of 520 nm.

DNA StainingNancy-520 can be used in a standard post-electrophoresis staining application for DNA on agarose gels. After the electrophoresis run, the gel is stained for 1 hour in the dark. The fluorescence image of the gel can be directly detected after a short rinse (see Figure 2).

Alternatively, Nancy-520 can also be used for pre-electrophoresis staining by simply adding the dye to the liquid agarose before pouring the gel. The gel can be imaged directly after the electrophoresis run without any further steps. Nancy-520 allows more detection possibilities than SYBR® Green. Detection is performed by illuminating the gel on a UV screen or using a Dark Reader®, and imaging the gel using a CCD-camera (e.g., KODAK® Gel-Logic-100) with a 535 nm or 590 nm band-pass filter, or a Polaroid® camera. Alternatively, a laser-scanner can be used (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.

23130 –9416 –6557 –4361 –

2322 –2027 –

564 –

Figure 2. DNA marker (Lambda DNA Hind III digest) at 2 different concentrations, was 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 camera. Right: Laser-scanner Fuji FLA-3000/λex 532 nm/λem 580 nm cut-off filter.

DNA Quantitation in SolutionNancy-520 can be used to determine dsDNA concentrations in solution. This application can be performed in a glass-bottomed 96 well plate, using known concentrations of dsDNA as a standard. Nancy-520 has a linear detection range between 0-2 μg/ml of DNA (see Figure 3).

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Figure 3. Quantitation of DNA (Lambda DNA Pst I Digest) in solution using Nancy-520, performed in a 96 well plate. Fluorescence was measured on a laser-scanner (FLA-3000, 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.

DNA Detection

Nancy-520 for Robust DNA Staining and Quantitation

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Nancy-520 and Related Products for DNA Analysis

Description Cat. No.

Nancy-520 01494-500UL

GenElute™ Agarose Spin Columns 56500-70EA

Agarose A4679-50GA4679-100GA4679-500G

Gel Loading Buffer G2526-5ML

Lambda DNA Hind III Digest D9780-20UGD9780-.1MGD9780-.5MGD9780-1MG

Lambda DNA Pst I Digest D1793-.1MGD1793-.5MGD1793-1MG

Tris-Borate-EDTA buffer T7527-1LT7527-4L

Tris Acetate-EDTA buffer T8280-100MLT8280-1L

DNA and RNA Intercalating FluorophoresNucleic 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 - 158550-10G158550-25G158550-100G

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 14530-100MG14530-500MG

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-F32670-25MG-F

Dimidium bromide 306 / 612 nm in 50 mM Tris pH 8.0 (DNA) 41785-250MG41785-1G

Ethidium bromide 530 / 600 nm in 50 mM phosphate buffer pH 7.0 (upon binding to DNA)

46065-1G46065-5G

Ethidium bromide solution 530 / 600 nm in 50 mM phosphate buffer pH 7.0 (upon binding to DNA)

46067-50ML-F46067-250ML-F

Ethidium homodimer 528 / 598 nm in TE buffer; DNA 46043-1MG-F46043-5MG-F

5-Methoxypsoralen - 275727-250MG275727-1G

Propidium iodide - P4170-10MGP4170-25MGP4170-100MGP4170-250MGP4170-500MGP4170-1G

Propidium iodide solution - P4864-10ML

Pyrene 338 / 375 nm in DMSO 82648-1G82648-10G

SYBR® Green II RNA gel stain - S9305-.5MLS9305-1ML

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New

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An increasing demand for red emitting fluorescent dyes, useful in life science applications such as antibody and protein labeling, prompted us to develop new improved long wavelength markers for use in the visible/near-infrared (NIR) region.

Tracy 645 and Tracy 652 (see Table 1) are proprietary dyes developed to meet the need for NIR fluorescent dyes. The Tracy dyes are:

� Able to retain chemical stability of the activated N-hydroxysuccinimide (NHS) ester under protein labeling conditions (basic pH, which prevents dye decomposition or hydrolysis that could result in poor labeling efficiency).

� Photostable.

� Soluble in aqueous labeling media as the activated NHS ester, reducing precipitation that leads to inefficient protein labeling.

� Flexible for use in typical protein labeling protocols.

The decrease in background fluorescence results from both a dramatic reduction of Rayleigh and Raman scattering by shifting to the longer wavelength excitation and from the diminished autofluorescence of sample impurities in this region.

Tracy 645 (free acid) Tracy 652 (free acid)

λ λex, max (639 nm) 280 nm λex, max (648 nm) 280 nm

absorption (PBS, pH 7)

0.343 1 μg/mL 0.495 50 μg/mL 0.3731.818 μg/mL

0.455 50 μg/mL

correction factor[4] 0.029 0.044

ε 222882 M-1cm-1 6437 M-1cm-1 181253 M-1cm-1 8028 M-1cm-1

λem,max

658nm (1 μg/mL, 0.1M phosphate buffer pH 7)

672nm (1 μg/mL, 0.1M phosphate 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.

Tracy 652 and Tracy 645 surpass conventional fluorescent labels, such as Cy® 5, in all of the above criteria (see Figure 1). Other, more modern labels of the corresponding type are also deficient in one or more of the above criteria.

600

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Tracy 652 (Cat. No. 38408)Cy5 (Amersham)

Figure 1. Photostability of Tracy 652 dye and Cy® 5.

Irradiation of each 1mM solutions in methanol in a white Pyrex flask by a 375 W white light lamp; distance from lamp to probe was 20 cm; probe maintained at room temperature; measured at λex,max and λem,max of each dye.

Investigation of optimal dye/protein (D/P) ratio of antibody conjugates of Tracy dyes showed a maximum emission intensity at ~4:1 D/P.

Secondary antibody conjugates with Tracy dyes are available for immunostaining applications observed by fluorescence microscopy (see Figure 2).

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).

New NIR Fluorescence Dyes

Tracy 645 and Tracy 652





New

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Additionally NTA (nitrilo-triacetate)-Tracy derivatives that selectively detect His-tagged proteins on solid surfaces such as gels or Western blots are provided by Sigma® Life Science (see Figure 3).

His

-Lad

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30 —

50 —

75 —

His-p38

500

ng

250

ng

100

ng

50 n

g

25 n

g

Figure 3. His-tagged p38-MAPK protein (500 ng–25 ng) was separated on a 4-20% Tris-Glycine SDS-PAGE gel. The gel was fixed overnight in 40% ethanol/10% acetic acid, washed in water, and incubated with Ni-NTA-Tracy 652 (1:1000) in the dark. The gel was washed and then imaged using a FLA-3000 Fuji® laser scanner with 633 nm excitation and a 675 nm emission filter. Ni-NTA-Tracy 652 (λex 652 nm, λem 677 nm) is excited in the red region of the spectrum.

The key advantages of this His-tagged protein detection method 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 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).

100–

45–

30–

12–

Mar

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500

250

250

100

100

50

50

5

5 [ng]

Protein 1: Atto550Protein 2: Tracy652

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

1. Wolfbeis, O.S., Fluorescence Spectroscopy: New Methods and Applications, Springer, Heidelberg, (1993).

2. Rettig, W. and B. Strehmel, B., Applied Fluorescence in Chemistry, Biology and Medicine, Springer, Heidelberg, (1999).

3. Mason, W.T. (ed.), Fluorescent and Luminescent Probes for Biological Activity, 2nd 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., 4, 105 (1993).

Life Science Innovations piques your interest with examples of new and emerging technologies put forth in a fresh, unique way that applies to your area of study.

Tailored for the life science researcher, BioFiles aligns our vast array of products within a relevant research topic.

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A Perfect Fit!

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Tracy DyesTracy 645 (activated) 8

corresponds for coupling to amines

42445-1MG 1 mg

Tracy 645 (free acid) 8

≥95.0% (HPCE)12670-1MG 1 mg

Tracy 652 (activated) 8

≥80% (coupling to amines)42454-1MG 1 mg

Tracy 652 (free acid) 8

≥95% (HPCE)38408-1MG 1 mg

Tracy-Conjugated AntibodiesAnti-Mouse IgG - Tracy 652 from goat antiserum 8

Tracy 652 - goat-Anti-mouse IgG 1 mg/mL protein

free of unconjugated dye; dye-to-protein ratio ≥2

42472-1G 1 g

Anti--Rabbit IgG - Tracy 645 from goat antiserum 8

Tracy 645- goat-Anti--rabbit IgG 1 mg/mL

free of unconjugated dye; dye-to-protein ratio >2

05557-1G 1 g

Anti-Rabbit IgG - Tracy 652 from goat antiserum 8

Tracy 652 - goat-anti-rabbit IgG 1 mg/mL protein

free of unconjugated dye; dye-to-protein ratio (molar) >2

43413-1G 1 g

Tracy-NTA ConjugatesNTA -Tracy 645 8

Tracy 645- Nitrilo tri acetic acid, com plex ed to Ni2+ - ion; Nα, Nα-bis(carboxy methyl)-L-lysine Nickel(II) com plex - Tracy 645 conjugate

18626-250UG 250 μg

NTA -Tracy 652 8

Tracy 652 - Nitrilo tri acetic acid, com plex ed to Ni2+ - ion; Nα, Nα-bis(carboxy methyl)-L-lysine Nickel(II) com plex - Tracy 652 conjugate

≥95% (HPCE)55183-250UG 250 μg

Tracy Protein Labeling KitsTracy Protein Labeling Kits provide an easy and reliable way to label purified proteins, enzymes and antibodies. The kits include a high quality Tracy dye and contain all components necessary to rapidly 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.

Tracy 645 Protein Labeling Kit 8

This kit contains sufficient amounts of reactive dye, buffers, and protein purification sets for performing five labeling reactions (1 mg protein each) and for the subsequent purification of the labeled protein.

91471-1KT 1 kit

Tracy 652 Protein Labeling Kit 8


75824-1KT 1 kit

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Detection Web Resource

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Car

bo

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Carbohydrate Detection

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 sigma.com/enzymeexplorer

Fluorescence image of rat testis. Seminiferous tubule with maturing germ cells. 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

Lectin from Phytolacca americana-Atto 647N conjugate 644 / 669 nm in PBS 03065-1MG

Lectin from Triticus vulgaris-Atto 488 conjugate 501 / 523 nm in PBS 16441-1MG

Lectin from Triticus vulgaris-Atto 532 conjugate 532 / 558 nm in PBS 68917-1MG2nd edition

Tools for Glycoproteomics and Glycomics

■ Glycan Labeling and Analysis

■ Glycoprotein Purification and Detection

■ Chemical and Enzymatic Deglycosylation

■ Enzymatic Synthesis and Degradation

Glycobiology Analysis Manual

JFY02060-4013200127

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Glycobiology Analysis Manual

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Carb

oh

ydrate D

etection

Fluorescent Labels for CarbohydratesDue 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 06627-25MG06627-100MG

7-Amino-4-methylcoumarin 365 / 440 nm in ethanol A9891-250MGA9891-500MGA9891-1GA9891-5G

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-F10609-100MG-F

9-Anthracenecarboxaldehyde - 278688-5G278688-25G278688-100G

Bis(2,4-dinitrophenyl) oxalate / 430 nm 75705-1G75705-5G

Bis(2,4,6-trichlorophenyl) oxalate / 430 nm 75707-5G75707-25G

4-Bromomethyl-7-methoxycoumarin 322 / 395 nm (after derivatization) 235202-250MG235202-1G235202-5G

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) 25455-1G25455-5G25455-25G

Dansyl chloride 337 / 492 nm in chloroform (after derivatization with hexylamine) 39220-1G-F39220-5G-F39220-50G-F

Dansylhydrazine 340 / 520 nm in ethanol 30434-250MG30434-1G30434-5G

7-(Diethylamino)coumarin-3-carbonyl azide 390 / 478 nm in methanol 31755-25MG

Diphenylborinic anhydride 366 / 475 nm (after derivatization) 358835-250MG358835-1G

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-F46985-500MG-F

4-Fluoro-7-nitrobenzofurazan 470 / 550 nm 47140-10MG47140-50MG

4-Hydrazino-7-nitrobenzofurazan 445 / 522 nm in chloroform (after derivatization with formaldehyde) 53892-50MG

Isoniazid 360 / 450 nm (thiol adduct) I3377-5GI3377-50GI3377-250G

Malonamide 367 / 445 nm (α-keto acid adduct) 129593-100G

4-Methoxybenzamidine 393 / 478 nm (after derivatization with glucose)393 / 478 nm

64785-100MG-F

4-(6-Methyl-2-benzothiazolyl)phenyl isocyanate 327 / 382 nm in methylene chloride (after derivatization with hexylamine)

65877-100MG65877-500MG

4,5-Methylenedioxy-1,2-phenylenediamine dihydrochloride 367 / 445 nm in 0.1 M phosphate pH 7.0 (after derivatization with pyruvate)

66807-10MG66807-50MG

1-Naphthaleneacetic anhydride 216 / 360 nm (polyamin adduct) 438952-250MG438952-1G

1-(2-Naphthoyl)imidazole 234 / 374 nm (after derivatization) 70684-500MG

o-Phenylenediamine 337 / 417 nm (after derivatization) P5412-50TABP5412-100TAB

o-Phenylenediamine - 78410-100G

2-Phenylphenol - P28263-500GP28263-2KG







































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Pro

tein

Det

ecti

on

Protein Detection

LUCY® Protein Stains

LUCY 506, LUCY 565, and LUCY 569 are fluorescence dyes for the sensitive detection of proteins on 1- or 2-dimensional 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 all 3 dyes. In addition, protein quantification in solution can be performed using LUCY 506 and LUCY 569.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

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.

LUCY 506: Blue line: excitation spectrum (maximum 506 nm); pink line: emission spectrum (maximum 520 nm).

LUCY 565: Orange line: excitation spectrum (maximum 565 nm); green line: emission spectrum (maximum 588 nm).

LUCY 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 stain is completed after 60 minutes, and detection is possible immediately thereafter.

� LUCY 565 is recommended if after staining, a Western blot transfer is to be performed from the same gel. Since staining is done under neutral, non-fixing conditions, the stained proteins may be transferred directly to the membrane. Using traditional dyes, 2 gels and, therefore, twice the amount of sample material would be needed.

� 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.

66 —

45 —

29 —

Mar

ker

250

100

75 50 25 10 5 3 1

66 —

45 —

29 —

Mar

ker

250

100

75 50 25 10 5 3 1

66 —

45 —

29 —

Mar

ker

250

100

75 50 25 10 5 3 1

66 —

45 —

29 —

Mar

ker

250

100

75 50 25 10 5 3 1

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, stained with LUCY 506. Bottom: 4-20% Tris-glycine gel, stained with LUCY 565. Both gels were imaged on a FLA-3000 laser scanner (Fuji®).

Depending on the chosen dye (see Figure 1), several devices can be used for the detection, e.g., illuminating the gel on a Dark-Reader® blue light transilluminator or a UV screen, and imaging the gel using a CCD- or Polaroid® camera. Alternatively, a laser-scanner can be employed, using the corresponding excitation and emission filter settings (see Figures 2 and 3).

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.





Protein

Detectio

n

% Int

m/z

1000 1500 2000 2500 3000 3500 4000

100908070

6050403020100

Figure 4. MALDI peptide mass fingerprint after in-gel-digest. 100 ng of E. coli β-galactosidase, separated by SDS-PAGE, was stained with LUCY 506 after band excision and trypsin-digest. MALDI spectra of extracted peptides, crystallized with HCCA and measured on a Shimadzu® Kratos CFR MALDI-MS instrument in reflectron mode.

Protein Quantification in SolutionLUCY® 506 and LUCY 569 can be also used to quantify proteins in solution. This application is designed for use in a 96-well format or in cuvettes. It is applicable for 2 different protein concentration ranges, 0-50 μg/ml and 10-1000 μg/ml and requires only buffer, SDS, and the LUCY dye (see Figure 5). Samples can be measured immediately after mixing.

2. High Range

0

2000

4000

6000

8000

10000

0 200 400 600 800 1000

µg/ml BSA

F/u

1. Low Range

0

2000

4000

6000

8000

10000

12000

0 10 20 30 40 50 60

µg/ml BSA

F/u

2. High Range

0

2000

4000

6000

8000

10000

0 200 400 600 800 1000

µg/ml BSA

F/u

1. Low Range

0

2000

4000

6000

8000

10000

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0 10 20 30 40 50 60

µg/ml BSA

F/u

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). Fluorescence detection was done on Fuji® FLA-3000 (λex 473 nm/λem 520 nm).

For more information about LUCY dyes and their applications in protein analysis, visit sigma.com/lucy_dyes

LUCY 506 solution 8

Lucy 506 is a fluorescent stain for protein electrophoresis, with high sensitivity and easy, fast and robust staining procedure for all kinds of SDS gels. Protein staining by Lucy 506 does not interfere with subsequent MALDI-MS.

Sensitivity of routine procedure is ca. 3 ng/band, with linear response between 3 and 1000 ng/band

Lucy 506 is provided as a 5000 x stock-solution in DMF (5 mg/ml)

λmax. ~495 nm (MeOH)

68721-500UL 500 μL

LUCY 565 solution

BioChemikaLucy 565 is a fluorescent stain for protein electrophoresis, with high sensitivity and easy, fast and robust staining procedure for all kinds of SDS gels. Lucy 565 is also suitable for neutral staining (e.g. for subsequent western blotting).

Protein staining by Lucy 565 does not interfere with subsequent MALDI-MS.



Lucy 565 is provided as a 5000 x stock-solution in DMSO (5 mg/ml)

43772-500UL-F 500 μL

LUCY 569 solution

BioChemika Lucy 569 is a fluorescent stain for protein electrophoresis, with good sensitivity and easy, fast and robust staining procedure for all kinds of SDS gels. Lucy 569 provides a broad linear dynamic range for protein quantification in electrophoresis. Protein staining by Lucy 569 does not interfere with subsequent MALDI-MS.



Lucy 569 is provided as a 5000 x stock-solution in DMSO (5 mg/ml)

41629-500UL-F 500 μL

LUCY Starter Kit 8

The LUCY Starter Kit contains three fluorescent protein stains for SDS-PAGE gels.

� LUCY 506 for high sensitivity staining with a detection limit of 3 ng protein per band. Staining is complete after 60 minutes and detection is possible immediately thereafter.

� LUCY 565 is recommended for staining SDS-PAGE gels prior to Western blot transfer. With traditional staining, 2 separate gels are required and therefore, double the amount of sample is necessary.

� LUCY 569 is particularly well suited for protein quantitation, displaying a broad, linear, dynamic range between 10–6,000 ng protein per band.

ComponentsLUCY 506 (Sigma 68721) 50 μLLUCY 565 (Fluka 43772) 50 μLLUCY 569 (Fluka 41629) 50 μL

51153-1KT 1 kit

Laemmli Lysis-buffer 8

non smellingin accordance for electrophoresis test

38733-1ML 1 mL

38733-5X2ML 5 × 2 mL

38733-5ML 5 mL

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Iso

elec

tric

Fo

cusi

ng

Isoelectric FocusingIsoelectric focusing (IEF) is a powerful analytical tool for the separation of proteins. In order to ensure the high performance of analysis, isoelectric point (pI) standards are needed. In addition to classical protein based standards, low molecular weight compounds have been developed and successfully examined in capillary IEF and IEF-gel electrophoresis. Gel electrophoresis, the common technology for IEF, minimizes convection and introduces an additional gel-sieving effect to separate proteins by size. However, it has several disadvantages such as lengthy analysis time, limited resolution, and difficulty in detection. These challenges have been largely overcome by the development of IPG-strips (immobilized pH gradients) for use in high-resolution pI-separation.

Capillary electrophoresis is a high-resolution approach to separate molecules, based on the pI point, that can be automated. Separation is carried out in fused-silica capillaries with internal diameters of 25-75 μm. The electrophoresis takes place in free solution and the capillary controls convection currents. After focusing is complete, the solutes are pumped out of the capillary. UV absorption is the most popular detection method for capillary IEF, but UV induced fluorescence emission is of interest for derivatized proteins. Dansyl chloride, fluorescamine, o-phthaldialdehyde, and coumarin moieties are used to increase detection sensitivity.

One of the most critical issues in isoelectric focusing, whether in capillary electrophoresis or slab gel, is standardization of results. For that purpose typical protein standards have been used which could show certain disadvantages:

� Limited stability of solutions

� Inadequate purity for application as a standard

� Lot-to-lot inconsistency

To overcome these problems, Sigma® Life Science introduces synthetic IEF-markers, which can be used for fluorescent detection. The fluorescent IEF-markers enable a much higher sensitivity than using UV detection, as is commonly used with slab gel electrophoresis. The maximum absorbances of the individual markers are between 308 and 350 nm. For fluorescence detection, an excitation wavelength of 310 nm is suggested (individual excitation maxima range between 310 to 400 nm); the emission maxima of the individual markers are between 410 and 500 nm (see Figure 1). An advantage in IEF gel electrophoresis with fluorescent IEF-markers is the ability to control the formation of a gradient without further staining (using UV illumination).

Minutes10 15

-0.006

-0.002

0.002

0.006

0.010

0.014

0.018

0.022

0.026

12

34

5

6

7

Peak Number

Au

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).




Isoelectric Fo

cusin

g

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 73938-1EA73938-200UL

Fluorescent IEF-Marker pI 6.6 solution 396 / 500 nm in 0.1 M phosphate pH 6.6 73376-1EA73376-200UL

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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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.


Buffer solution HPCE pH 2.5, BioChemika, for HPCE, 50 mM sodium phosphate

82635-50ML82635-100ML


82578-50ML82578-100ML82578-500ML

Buffer solution HPCE pH 3.0, BioChemika, for HPCE, for the detection of alkali and alkaline earth metals and amines

82621-50ML82621-100ML

Buffer solution HPCE pH 3.0, BioChemika, for HPCE, 20 mM sodium citrate

82582-50ML82582-100ML

Buffer solution HPCE pH 3.0, BioChemika, for HPCE, 150 mM potassium phosphate

82622-100ML82622-500ML


82583-50ML82583-100ML


82584-50ML82584-100ML82584-500ML


82585-50ML82585-100ML82585-500ML


82586-50ML82586-100ML82586-500ML


82588-50ML82588-100ML82588-500ML


82589-50ML82589-100ML82589-500ML


82591-50ML82591-100ML82591-500ML


82637-50ML82637-100ML


82636-50ML82636-100ML



82592-50ML82592-100ML82592-500ML

Buffer solution HPCE pH 7.7, BioChemika, for HPCE, for the detection of inorganic and low molecular weight organic anions

82619-50ML82619-100ML

Buffer solution HPCE pH 8.0, BioChemika, for HPCE, 20 mM sodium tetraborate

82594-50ML82594-100ML

Buffer solution HPCE pH 8.0, BioChemika, for HPCE, 100 mM sodium borate

82634-100ML82634-500ML

Buffer solution HPCE pH 8.0, BioChemika, for HPCE, 50 mM sodium borate

82633-50ML82633-100ML


82593-50ML82593-100ML

Buffer solution HPCE pH 9.0, BioChemika, for HPCE, 20 mM sodium tetraborate

82604-50ML82604-100ML82604-500ML


82603-50ML82603-100ML


82605-50ML82605-100ML82605-500ML

Buffer solution HPCE pH 10.0, BioChemika, for HPCE, 20 mM CAPS [3-(cyclohexylamino)-1-propane sulfonic acid]

82606-50ML82606-100ML82606-500ML


82607-50ML82607-100ML82607-500ML

Buffer solution HPCE pH 11.0, BioChemika, for HPCE, 20 mM glycine-NaOH

82617-50ML82617-100ML


82608-50ML82608-100ML82608-500ML








































































Detectio

n K

its

Detection Kits

Ampliflu Red Western Blot Kit

Classical immunoblotting for protein detection on a membrane is often based on horseradish peroxidase (HRP) labeled secondary antibodies. Peroxidase can be detected directly using colorimetric substrates, or chemi luminescence-based substrates with CCD camera or X-ray film detection.

The new Ampliflu Red Western Blot Kit contains a peroxidase 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 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 accumulation is necessary as it is for chemiluminescent signals. The use of Ampliflu Red is suitable for protein quantities between 1 ng and 1 μg per band of protein.

In order to receive an optimal signal to background ratio, it is strongly recommended to use a low-fluorescence PVDF-membrane for the Western blot transfer and a 5% BSA blocking solution.

ExcitationFluorescence

Imaging

Ampliflu Red Resorufin

sec. antibody–HRP

prim. antibody

Membrane

Protein

Figure 1. Schematic overview for the use of Ampliflu Red. A protein is immobilized by western blot transfer after SDS-PAGE. The protein is bound with a primary antibody and followed by a secondary antibody carrying a horseradish peroxidase (HRP) label. The membrane is incubated in a solution containing Ampliflu Red and hydrogen peroxide. Ampliflu Red is converted into resorufin by the action of HRP. The resultant fluorescent signal can be detected at the excitation and emission parameters of resorufin (λex = 571 nm, λem = 585 nm).

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40 n

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30 n

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20 n

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10 n

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5 ng

1 ng

FLAG-BAP protein

Figure 2. Detection of FLAG-BAP™ protein (50 ng – 1 ng) by immunoblotting using the Ampliflu Red Western blot Kit on Immobilon®-FL PVDF-Membrane. The membrane was blocked using 5% BSA in PBS solution and then incubated with monoclonal 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 (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.

Blocking with 5 % BSA in PBS(1 h-overnight)

Short rinse with PBST

Incubate with primary antibody (2-3 h)

Wash with PBST (3 x 5 min)

Incubate with secondary antibody-HRP (1 h)

Wash with PBST (3 x 5 min)

Short rinse with PBS

Fluorescent imaging

Ampliflu Red Western Blot Kit

5 min incubation with this mixture: • 1880 µl PBS • 20 µl Ampliflu Red solution • 100 µl H2O2 solution

Figure 3. Protocol for the use of Ampliflu Red after SDS-PAGE and Western blot transfer. For membranes in the mini-format, the blocking and washing steps were done in a volume of 50 mL, whereas, the antibody incubations were done in 25 mL. The Ampliflu Red incubation mixture in PBS has a total volume of 2 mL. PBST contains 0.1% TWEEN® 20.


Ampliflu Red Western Blot Kit 79454-1KT

TWEEN® 20 P5927-100MLP5927-500ML

Immobilon®-FL PVDF membrane 05317-10EA

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Det

ecti

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Kit

s

Nitrite/Nitrate Assay Kit

The Nitrite/Nitrate Assay Kit can be used to detect nitrite (NO2-) and

nitrate (NO3-). The detection of NO2

- is determined by direct reaction with 2,3-diaminonaphthalene (DAN) (see Figure 1). Fluorescence of the resulting naphthotriazole can then be measured. The excitation maximum of naphthotriazole is 360-365 nm, with an emission maximum at 410-425 nm. However, in order to reduce the fluorescence background of 2,3-diaminonaphthalene and 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 also be done using cuvettes, depending on available equipment.

NO3- can only be detected after enzymatic conversion to NO2

-. Subsequent subtraction of NO2

- yields the NO3- concentration.

The optimal detection range is 1-10 μM for [NO2- + NO3

-].

NO3- + NADPH + H+ enzyme NO2

- + NADP+ + H2O

NO2- + DAN + H+ Naphthotriazole (fluorescent) + 2 H2O

Calibration curves

0

50

100

150

200

250

0 2 4 6 8 10

µM

F/u

Nitrite

Nitrate

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.

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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n Full range of progressive technologies for biomarker discoveryn Proteomic standards, calibrants, and markersn Intuitive design tools for peptide libraries

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Sensor Dyes

Fluorogenic NO Bioimaging Probes

In 1998 R.F. Furchgott, L.J. Ignarro, and F. Murad received the Nobel Prize in Physiology and Medicine for their discoveries concerning nitric oxide as a signaling molecule in the cardiovascular system. This indicates the importance the scientific community has placed on the role of nitric oxide (NO) as a signal transporter in neurons, endothelial cells and in the immune system. NO has been implicated in vasodilation,1 neurotransmission,2 cytotoxicity, immune response,3 and inflammation.4-6

Within cells, nitric oxide synthase (NOS) catalyzes the conversion of arginine to citrulline plus NO in the presence of molecular oxygen, tetrahydrobiopterin, NADPH, and flavin cofactors. Nε-hydroxy-L-arginine is an intermediate in this reaction. NO has an in vivo half-life of only a few seconds,7 however, since it is soluble in both aqueous and lipophilic media, it readily diffuses through the cytoplasm and across cell membranes. NO has effects on neuronal transmission as well as synaptic plasticity in the central nervous system. NO activates guanylate cyclase in the vasculature, thus, increasing the synthesis of cGMP, which in turn induces smooth muscle relaxation and vasodilation. NO toxicity is related to its ability to combine with superoxide anions to form peroxynitrite, an oxidizing free radical that can damage DNA and other cellular constituents.

The NO molecule is unstable and occurs in very low concentrations in biological systems. In order to detect it in real time, methods with high sensitivities and specificities are required. Fluorescence microscopy seems to be the method of choice, provided suitable NO probes are available. This high spatial-temporal resolution technique allows for reliable imaging of in situ NO concentrations.

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.

O

O

O

AcO OAc

NH2

H2N

O

O

O

HO OH

NH2

H2N

O

O

O

HO OH

NHN

N

intracellularesterases NO / O2

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.

Naphthalenes2,3-Diaminonaphthalene (DAN) was established as a sensitive probe to measure NO-concentration in vitro and under physiological conditions.11 The respective 2,3-naphthotriazole (NAT) was detected in the low nM concentration range. DAN, however, proved to be ill-suited to NO detection in cells due to rapid leakage through cell membranes after loading. High background fluorescence in biological matrices was also experienced due to its rather short excitation and emission wavelengths.

Therefore, DAN-1 and its cell membrane permeable precursor DAN-1-EE were developed.12 DAN-1 is formed by the hydrolysis of DAN-1-EE by cytosolic esterases. DAN-1-T (see Figure 2), the fluorescent reaction product of DAN-1 with NO, showed considerably improved leakage behavior.

0

100

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300

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700

0 10 20 30 40 50 60 70

[min]

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a.u

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Figure 2. Time course of formation of fluorescent DAN-1-T from DAN-1 upon addition of final concentration of 5 mM (green line) or 50 mM (red line) respectively of a NO forming agent (NONOate)[12].



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XanthenesFluorescein and rhodamine-based probes have been developed in order to eliminate some drawbacks of naphthalene-based NO probes, such as cytotoxicity, strong autofluorescence, a small extinction coefficient, poor solubility in neutral buffer and short excitation/emisson wavelengths.10,13-16

Limit of detection (LOD) of NO by DAF-2 is 5 nM in vitro, in the absence of absorbing side products. Oxidized forms of NO such as NO2- and NO3-, as well as reactive oxygen species such as O2

·-, H2O2, and ONOO- do not react with DAF-2 to give a fluorescent product. Therefore, under physiological conditions fluorescent DAF-2 T is only formed in the presence of NO.

A fluorescent response was detected when cultured, smooth aortic rat muscle cells loaded with DAF-2-DA were viewed using a confocal laser scanning microscope in the presence of endotoxins and cytokines. The addition of L-arginine resulted in a sharp increase of the fluorescence signal, whereas supplementary addition of N-monomethyl-L-arginine (L-NMMA) resulted in no signal increase, clearly demonstating the inhibition of NO-synthase.

The fluorescein type probe (DAF-2) shows a bright fluorescence signal; while the rhodamine type probes (DAR-1 and DAR-2) excel with high photostability and their applicability over a broad pH range (see Table 1, Table 2 and Figure 3).

probeλabs., max. [nm] ε [x104 M-1cm-1] λem., max. [nm] rel. quantum yields

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

DAR-2 549 552 10 7.1 571 0.006 0.34

Table 1. Spectral properties of DAF and DAR probesa)b)

a) Data from reference.11

b) Data at 20 °C in 0.1 M phosphate buffer, pH 7.4; quantum yields derived by comparison with known quantum yield of rhodamin B in ethanol.

DAF’s DAR’s

λex [nm] ~490 ~550

λem [nm] ~515 (green) ~575 (red)

suitable pH range > pH 6 > pH 4

detection limit for NO ~5 nM (DAF-2, in vitro) ~10 nM (DAR-M, in vitro)

photostabilityb) photobleached more than 70% not photobleached

distribution nucleus/cytosol equal cytosol > nucleus

Table 2. General Properties of DAF and DAR probesa)b)

a) Data from references.10-12

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

0

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800

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2 3 4 5 6 7 8 9 10 11

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Figure 3. pH dependence of fluorescence intensity (a.u.) of DAF-2 (green), DAR-1 (orange) and DAR-2 (pink)[11].

References

1. Palmer, R.M., et al., Nature, 327, 524 (1987).2. J. Garthwaite, J., et al., Nature, 336, 385 (1988).3. Marletta, M.A., et al., Biochemistry, 27, 8706 (1988).4. Stuehr, D.J., Ann. Rev. Pharmacol. Toxicol., 37, 339 (1997).5. Marletta, M.A., et al., Curr. Opin. Chem. Biol., 2, 656 (1998).6. Geller, D.A. and Billiar, T.R., Cancer Metastasis Rev., 17, 7 (1998).7. Mordvintcev, P., et al., Anal. Biochem., 199, 142 (1991).8. Misko, T.P., et al., Anal. Biochem., 214, 11 (1993).9. Miles, A.M., et al., Methods Enzymol., 268, 105 (1996).10. Kojima, H., et al., Anal. Chem., 70, 2446 (1998).11. Andrew. P.J., et al., FEBS Lett., 408, 319 (1997).12. Kojima, H., et al., Biol. Pharm. Bull., 20, 1229 (1997).13. Kojima, H., et al., Tetrahedron Lett,. 41, 69 (2000).14. Kojima, H., et al., Anal. Chem., 73, 1967 (2001).15. Kojima, H., et al., Chem. Pharm. Bull., 46, 373 (1998).16. Nakatsubo, N., et al., FEBS Lett., 427, 263 (1998).

Fluorogenic Probes

Name Cat. No.

DAR-1 09014-1MG-F

DAR-2 08715-1MG-F08715-5MG-F

4,5-Diaminofluorescein D224-1MG

5,6-Diaminofluorescein diacetate 50277-1MG-F

4,5-Diaminofluorescein diacetate D2813-1MG

2,3-Diaminonaphthalene 88461-25MG-F88461-100MG-F

Ethyl 4-[(3-amino-2-naphthyl)aminomethyl]benzoate hydrochloride

16513-1MG-F16513-10MG-F

4-[(1-Naphtho[2,3-d]triazol-1-yl)methyl]benzoic acid 36495-1MG-F36495-10MG-F

















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Convergence of Life Science & Materials Science Products and Expertise to Create:

The new Biomaterials resource is the simplest tool for finding information and products for Biomaterials Research. Visit sigma-aldrich.com/biomaterials

Lipids

Microparticles

Fluorescent Labels

Biomaterialssigma-aldrich.com/biomaterials

Polymers

Structural Proteins

Cell Culture

O

O

CH3n

Peptides, Custom DNA Synthesis and More

Additional Reagents for NO Bioimaging

Name Cat. No.

3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene A5581-10MGA5581-50MG

3-Bromo-7-nitroindazole B2050-10MG

Cyclosporin A 30024-25MG30024-100MG

Diethylamine NONOate diethylammonium salt D5431-50MG

Diethylamine NONOate sodium salt hydrate D184-25MGD184-50MG

3-Ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene E3145-10MG

(±)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide E2895-10MG

(±)-(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide

E3020-10MG

Ketorolac tris salt K1136-1GK1136-5G

MAHMA NONOate M1555-50MG

3-Morpholinosydnonimine hydrochloride M5793-25MGM5793-100MG

Nitric Oxide Synthase Detection Kit, Isotopic NOS1-1KT

Name Cat. No.

S-Nitroso-N-acetyl-DL-penicillamine N3398-25MGN3398-50MG

NOC-5 A5456-10MG

Sodium nitroprusside dihydrate 71778-25G71778-100G

Spermidine trihydrochloride 85580-5G85580-25G

Spermine S3256-1GS3256-5GS3256-25G

Spermine tetrahydrochloride S2876-1GS2876-5GS2876-10GS2876-25GS2876-100G

Spermine–Nitric oxide complex hydrate S150-25MG

Streptozocin S0130-50MGS0130-100MGS0130-500MGS0130-1GS0130-5G

Sulfo NONOate disodium salt S8432-50MG


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Chelators and Ion ProbesIons 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 tetrakis(acetoxymethyl) ester

- 40843-10MG-F

1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester

- 16609-10MG-F

1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid - A4926-250MGA4926-1GA4926-5G

1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrasodium salt - 14513-250MG14513-1G

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 - C0875-5GC0875-10GC0875-25G

Calcein disodium salt - 21030-1G-F21030-5G-F21030-100G-F

Calcein-AM 496 / 516 nm in 0.1 M Tris pH 8.0, esterase; Ca2+ 17783-1MG17783-5MG

Calcein AM solution - C1359-100UL

Calcein Blue - M1255-1GM1255-10G

2,3-Diaminonaphthalene 364 / 406 nm in 0.5 M Na carbonate pH 10.0 (after derivatization with NaNO2 in 0.1 M HCl)

88461-25MG-F88461-100MG-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-F46393-5MG-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 - I3261-250UGI3261-1MG

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 - 72543-1MG72543-5MG

PBFI-AM 370 / 540 nm in methanol 76275-1MG

29H,31H-Phthalocyanine - 253103-1G253103-5G

1,3,6,8-Pyrenetetrasulfonic acid tetrasodium salt hydrate - 82658-1G

2,6-Pyridinedicarboxylic acid - P63808-25GP63808-100GP63808-500G

Quin-2 332 / 493 nm in 0.1 M Tris pH 8.0, 10 mM Ca2+ 08520-50MG

Quin 2-AM - Q4875-10MGQ4875-50MG

SBFI-AM - S1148-.1MGS1148-1MG

5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) - 323497-100MG323497-250MG

4,4,4-Trifluoro-1-(2-naphthyl)-1,3-butanedione - 343633-5G343633-25G






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pH IndicatorspH 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 - A38401-5GA38401-25G

8-Anilino-1-naphthalenesulfonic acid ammonium salt - 10417-5G-F10417-25G-F10417-100G-F

8-Anilino-1-naphthalenesulfonic acid hemimagnesium salt hydrate - A5144-5GA5144-25G

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 17283-5MG17283-25MG

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-F21877-5G-F

5-Carboxyfluorescein diacetate - C4916-25MGC4916-100MG

5(6)-Carboxyfluorescein diacetate 492 / 517 nm in 0.1 M Tris pH 8.0 (esterase) 21879-25MG-F21879-100MG-F

6-Carboxyfluorescein diacetate - C5041-25MGC5041-100MG

5(6)-Carboxyfluorescein N-hydroxysuccinimide ester 492 / 517 nm in DMF 21878-25MG-F21878-100MG-F

5(6)-Carboxynaphthofluorescein 598 / 668 nm (basic)512 / 567 nm (acid/neutral)

21932-25MG-F21932-100MG-F

Chromoionophore I - 27086-10MG-F27086-100MG-F

3-Cyanoumbelliferone 408 / 450 nm in methanol 28605-100MG28605-500MG

3,6-Diacetoxyphthalonitrile - D0679-5MG

6,8-Dihydroxy-1,3-pyrenedisulfonic acid disodium salt 458 / 498 nm in 0.1 M Tris pH 8.0405 / 456 nm in 0.1 M citrate pH 3.0

37920-100MG37920-500MG

N,N-Dimethyl-6-propionyl-2-naphthylamine 361 / 498 nm in methanol 41525-25MG41525-100MG

6-Dodecanoyl-N,N-dimethyl-2-naphthylamine 366 / 497 nm in methanol 40227-100MG

Eosin diacetate - 45244-1G45244-5G

Fluorescein diacetate - F7378-5GF7378-10GF7378-25GF7378-100G

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-F55156-100MG-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 55627-100MG55627-500MG






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Sen

sor

Dye

s


7-Hydroxy-N-octadecylcoumarin-3-carboxamide 359 / 406 nm in chloroform/DMSO (1:1) 56059-100MG-F56059-500MG-F

8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt - H1529-1G

Naphthofluorescein 594 / 663 nm in 0.1 M Tris pH 9.0 70420-100MG70420-500MG

8-Octadecyloxypyrene-1,3,6-trisulfonic acid trisodium salt 404 / 434 nm in H2O 74758-50MG

2-Phenylphenol - P28263-500GP28263-2KG

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 - T9792-250MGT9792-1G

Liphophilic and Membrane ProbesFluorescent 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 07985-1G07985-5G

7-Amino-4-methylcoumarin 365 / 440 nm in ethanol A9891-250MGA9891-500MGA9891-1GA9891-5G

7-Amino-4-methyl-3-coumarinylacetic acid 350 / 433 nm in methanol 08445-100MG08445-500MG

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 - 278688-5G278688-25G278688-100G

2-Anthracenesulfonyl chloride 382 / 421 nm in acetonitrile (after derivatization with hexylamine) 06479-100MG-F

4-Bromomethyl-7-methoxycoumarin 322 / 395 nm (after derivatization) 235202-250MG235202-1G235202-5G

5(6)-Carboxy-X-rhodamine 570 / 595 nm in 0.1 M Tris pH 8.0 21965-25MG-F21965-100MG-F

5(6)-Carboxytetramethylrhodamine 543 / 570 nm in methanol 21953-100MG-F21953-500MG-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 - D0531-100MGD0531-500MGD0531-1G

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 36801-25MG36801-100MG

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 - 318426-250MG318426-1G

pH Indicators, continued





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Senso

r Dyes


N-(7-Dimethylamino-4-methyl-3-coumarinyl)maleimide 398 / 482 nm in 0.1 M phosphate pH 7.0 (after derivatization with 2-mercaptoethanol)

39145-10MG

N,N′-Dimethyl-9,9′-biacridinium dinitrate - M8010-500MGM8010-1GM8010-5G

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 - D208000-1GD208000-5G

Diphenylmaleic anhydride 284 / 506 nm (after derivatization with hexylamine) 43095-1G43095-5G

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 - F7250-50MGF7250-100MGF7250-250MGF7250-500MGF7250-1GF7250-5G

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) 63184-10MG63184-25MG

Merocyanine 540 - 323756-100MG323756-250MG

7-Methoxycoumarin-3-carboxylic acid 330 / 402 nm in 0.1 M Tris pH 9.0 64948-100MG64948-500MG

10-Methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate - 68617-25MG68617-100MG

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-F50726-5MG-F

6-(7-Nitrobenzofurazan-4-ylamino)hexanoic acid 466 / 535 nm in methanol 72964-100MG72964-500MG

1-Pyrenebutyric acid - 257354-1G257354-5G

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 - P7908-100MGP7908-500MGP7908-1G

Rhodamine 123 - 83702-10MG83702-50MG

Rhodamine B isothiocyanate - R1755-100MGR1755-500MGR1755-1G

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 87918-10MG87918-50MG

Tetramethylrhodamine methyl ester perchlorate - T5428-25MG

6-(p-Toluidino)-2-naphthalenesulfonyl chloride 381 / in methanol 04076-50MG04076-250MG

N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenylammonium p-toluenesulfonate

355 / 430 nm in methanol 43060-25MG-F


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New

Pro

du

ct C

orn

er

New Product CornerAtto 488 Protein Labeling Kit 8

This kit contains sufficient amounts of reactive dye, buffers and protein purification sets for performing five labeling reactions (1mg protein each) and for the subsequent purification of the labeled protein.

38371-1KT 1 kit

Atto 550 Protein Labeling Kit 8

This kit contains sufficient amounts of reactive dye, buffers and protein purification sets for performing five labeling reactions (1 mg protein each) and for the subsequent purification of the labeled protein.

51146-1KT 1 kit



68616-1KT 1 kit

Atto 647N Protein Labeling Kit 8


76508-1KT 1 kit



51253-1KT 1 kit



73919-1KT 1 kit

Rhodol 8

3′-Amino-6′-hydroxy-fluor an [3086-44-0] C20H13NO4 FW 331.32

BioChemika, ≥85% (HPCE)Rhodol is a strongly fluorescent dye. It is a structural hybrid of fluorescein and rhodamine.

67806-5MG 5 mg

N-(5-Fluor esceinyl)male imide 8

5-Male imido-fluor escein [75350-46-8] C24H13NO7 FW 427.36

≥90% (HPLC)corresponds for coupling to thiols

38132-25MG 25 mg

N-(4-Methyl umbelli feryl)male imide7-Male imido-4-methyl cou marin [211565-47-8] C14H9NO4 FW 255.23corresponds for coupling to thiols

92333-25MG 25 mg

CYPMPO 8

5-(2,2-Dimethyl-1,3-pro poxy cyclo phos phor yl)-5-methyl-1-pyrroline N-oxide; 2-(5,5-Dimethyl-2-oxo-2λ5-[1,3,2]dioxaphos phinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide [934182-09-9] C10H18NO4P FW 247.23

≥97% (HPLC)CYPMPO is designed for radical detection. It is a free radical spin trap with excellent trapping capabilites toward hydroxyl and superoxide radicals in biological and chemical systems.

01872-1MG 1 mg

Atto 633

BioChemika, for fluorescence18620-1MG 1 mg

N-Bio tin yl-3,6,9-tri oxa undec ane-1,11-di amine trifluoroacetate salt solutionN-{2-[2-(2-(2-Amino ethoxy)ethoxy)ethoxy]ethyl}bio tin amide trifluoroacetate salt [945462-84-0] C18H34N4O5S · C2HF3O2 FW 532.57

≥95.0% (TLC), 25 mg/mL in DMSONucleophilic biotinylation reagent for the attachment of cell surface glycosides to ELISA-type surfaces via a hydrophilic spacer/linker.

38801-25MG-F 25 mg

38801-100MG-F 100 mg

N-Bio tin yl-ethylene di amine trifluoroacetate saltN-(2-Amino ethyl)bio tin amide trifluoroacetate saltC12H22N4O2S · C2HF3O2 FW 400.42

≥96.5% (HPLC)Nucleophilic biotinylation reagent for the conjugation of various substrates and their identification and quantification using biotin-binding fluorescent labels

08599-100MG 100 mg

08599-500MG 500 mg

Nε-(N-(+)-Bio tin yl-6-amino hexa noyl)-Nα,Nα-bis(carboxy methyl)-L-lysine tripotassium saltBio tin-X-NTA [856661-92-2] C26H40K3N5O9S FW 715.98

BioChemika, ≥98.0% (TLC)Hetero-bifunctional linker for His-tagged proteins on one side and (strept)avidin conjugates

51410-5MG 5 mg

51410-25MG 25 mg

Atto 655 Phalloidin 8

BioChemika, ≥95.0% (HPCE, sum of isomers)Fluorescent labeled phalloidin is used for the specific labeling of microtubuli.

18846-10NMOL 10 nmol

Ovalbumin-Atto 488 conjugate 8

Fluorescent labeled ovalbumin is used as endocytic tracer.

free of unconjugated dye

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

























Bo

oks

BooksGreen Fluorescent Protein: Properties, Applications and Protocols, 2nd ed.

This text tackles both theory and practice, offering numerous case studies, examples, illustrations, and troubleshooting tips. It examines how Ps are tailored for specific systems and used to maximize expression, and how variants are generated with altered properties. The text also explores new ways to use FPs and methods for enhancing detection in a variety of organisms and cell types. This updated and expanded edition places emphasis on the rise of FPs and their applications in industry and biosensor research. It provides new chapters on FPs, biosensors, and advances in the use of FPs in the biotechnology and pharmaceutical industries, detailed information about protocols using FPs, and three-dimensional structure, and molecular biology and mutation of FPs.

Z705217-1EA 1 ea

Luminescence Biotechnology: Instruments and Applications

The dangers inherent in radioactivity-based methods have caused a shift towards luminescence measurements and visualization techniques. This up-to-date review covers the fundamentals of different luminescence-based assay systems, calculation methods, and instruments through the spectrum of applications and research advances. Topics include gene and protein assays, oxidative stress and tissue aging, applications of luminescent microspheres, and proton image analysis. This book clearly identifies the advantages of luminescence over other assay techniques, discusses its potential pitfalls, and illustrates the broad range of its utility.

L3165-1EA 1 ea

Molecular Fluorescence: Principles and Applications

Fluorescence spectroscopy is an important tool of investigation in many areas. Its advantage is extremely high sensitivity and selectivity - even single molecules can be detected - and it achieves a high spatial resolution and time resolution in combination with microscopic techniques or laser techniques, respectively. In biochemistry and molecular genetics, fluorescence spectroscopy has become a dominating technique. Together with the latest imaging techniques, fluorescence spectroscopy allows a real-time observation of the dynamics of intact biological systems with an unprecedented resolution.

M0566-1EA 1 ea

Principles of Fluorescence Spectroscopy 3rd ed.

The third edition of the established classic text reference, will enhance upon the earlier editions’ successes. It will maintain the emphasis on basics, while updating the examples to include recent results from the literature. This edition also includes new chapters on single molecule detection, fluorescence correlation spectroscopy, novel probes and radiative decay engineering. The full color text features the following: - Problem sets following every chapter - Glossaries of commonly used acronyms and mathematical symbols - Appendices containing a list of recommended books which expand on various specialized topics - Sections describing advanced topics will indicate as such, to allow these sections to be skipped in an introductory course, allowing the text to be used for classes of different levels.

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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LEM71061-508360

0029





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innovative solutions for fluorescent and chemiluminescent applications - biofiles issue 4.1

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