biofiles - solutions for detection

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Detection

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INTRODUCING

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IntroductionFluorescent techniques are widespread and fast-growing analytical methods used in life science. They allow sensitive and selective investigation of biological processes, diagnostic screening, kinetics and conformational studies. Research is evolving from identification of a large number of target molecules to isolation and investigation at the level of a single molecule. Key innovations for microscopy and nanoscopy in the last decade have been:

• Confocal laser scanning microscopy (CLSM)

• Fluorescence correlation spectroscopy (FCS)

• Total internal reflection of fluorescence (TIRF)

• Stimulated emission depletion (STED) microscopy

• Ground State Depletion (GSD) Microscopy

• Spectral precision distance microscopy (SPDM)

• Scanning nearfield optical microscopy (SNOM)

• Fluorescence photoactivation localization microscopy (FPALM)

• Stochastic optical reconstitution microscopy (STORM)

These super-resolution microscopic and spectroscopic techniques allow a tremendous increase in lateral resolution and enable scientists to have a new perspective of cellular studies. For example, STED microscopy, the revolutionary technique invented by Professor Stefan Hell, enables a microscopic resolution limit below the theoretical limit of resolution and permits more detailed studies in cellular processes.Sigma offers a comprehensive selection of reagents for superior application results including the fluorescent Atto dyes series, CF-Dyes and also anti-Stokes Sunstones® probes developed by Intelligent Material. Sigma has recently launched the new protein detection assay Duolink® developed by Olink Bioscience that allows precise detection and quantification of proteins, protein interactions and protein modifications in fixed cells and tissue samples.

Table of Contents

Duolink® Using PLA® Technology 4-5

Prestige Antibodies® 6

Sunstone® Luminescent UCP Nanocrystals 7-10

CF™ Dyes 11

Mix-n-Stain™ Antibody Labeling Kit 12

Fluorescent Dye Comparison 13-14

Atto™ Dyes 15-18

Abberior® Dyes 19-20

Spectral Fluorescence Standard Kit 21-22

Accelerate the Signaling Pathway Discovery ProcessDetect, quantify and determine cell localization of a specific protein complex in the same experiment. Duolink, based on in situ PLA, which is a proximity ligation assay, enables you to visualize protein interactions in fixed cells and tissue samples, all while under endogenous protein expression.

• Visualize individual interactions without having to overexpress proteins

• Gain high specificity with dual binding of primary antibodies

• Single molecule sensitivity due to signal amplification

• Analyze using standard immunofluorescence instruments

The Duolink kit series of optimized, simple to use reagents, allows the user to combine any pair of immunofluorescence or immunohistochemistry validated antibodies for direct in-cell detection of protein interaction events. Duolink read-out is performed either with a fluorescent label for fluorescence microscopy or HRP for brightfield detection.The resulting distinct spots are derived from individual protein interaction events, which are visualized using a standard microscope.

Preparation

STEP 1. Fix cells or tissues onto microscope slide or microplate.

STEP 2. Wash and add two primary antibodies.

STEP 3. Wash and add the PLUS and MINUS PLA probes.

Detection

STEP 4. Wash and add Ligation solution.

STEP 5. Wash and add Amplification solution.

STEP 6. Review and capture images.

Analysis

STEP 7. Single protein interactions visualized using fluorescence and brightfield, respectively.

STEP 8. Obtain objective quantification using Duolink Image Tool.

STEP 9. Data analysis.

Cells or tissue deposited on slides or in microplates are fixed (Step 1) to preserve activation status and transient interactions. Validated primary antibodies for the targets are added (Step 2) followed by binding of the PLA probes (Step 3). Hybridization of the oligonucleotide arms of the PLA probes will create a template for rolling circle amplification (RCA) only when the epitopes of the target proteins are in close proximity (<40 nm) (Step 4), followed by amplification and labeling of the RCA product by detection probes (Step 5). The result can be detected and visualized by standard microscopy (Step 5, 6 and 7).

Discover your pathway at

sigma.com/duolink

Duolink® Using PLA® Technology

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Cat. No. Product Description Size

PLA Probes PLUSDUO92001-30RXN Duolink In Situ PLA probe anti-Mouse PLUS 30

DUO92001-100RXN Duolink In Situ PLA probe anti-Mouse PLUS 100

DUO92002-30RXN Duolink In Situ PLA probe anti-Rabbit PLUS 30

DUO92002-100RXN Duolink In Situ PLA probe anti-Rabbit PLUS 100

DUO92003-30RXN Duolink In Situ PLA probe anti-Goat PLUS 30

DUO92003-100RXN Duolink In Situ PLA probe anti-Goat PLUS 100

PLA Probes MINUSDUO92004-30RXN Duolink In Situ PLA probe anti-Mouse MINUS 30

DUO92004-100RXN Duolink In Situ PLA probe anti-Mouse MINUS 100

DUO92005-30RXN Duolink In Situ PLA probe anti-Rabbit MINUS 30

DUO92005-100RXN Duolink In Situ PLA probe anti-Rabbit MINUS 100

DUO92006-30RXN Duolink In Situ PLA probe anti-Goat MINUS 30

DUO92006-100RXN Duolink In Situ PLA probe anti-Goat MINUS 100

ProbemakerDUO92009-1KT Duolink In Situ Probemaker PLUS 20 μg

DUO92010-1KT Duolink In Situ Probemaker MINUS 20 μg

Detection Reagents Orange

DUO92007-30RXN Duolink In Situ Detection Reagents Orange1 30

DUO92007-100RXN Duolink In Situ Detection Reagents Orange1 100

Detection Reagents RedDUO92008-30RXN Duolink In Situ Detection Reagents Red2 30

DUO92008-100RXN Duolink In Situ Detection Reagents Red2 100

Detection Reagents Far RedDUO92013-30RXN Duolink In Situ Detection Reagents Far Red3 30

DUO92013-100RXN Duolink In Situ Detection Reagents Far Red3 100

Cat. No. Product Description Size

Detection Reagents GreenDUO92014-30RXN Duolink In Situ Detection Reagents Green4 30

DUO92014-100RXN Duolink In Situ Detection Reagents Green4 100

Detection Reagents BrightfieldDUO92012-30RXN Duolink In Situ Detection Reagents Brightfield5

DUO92012-100RXN Duolink In Situ Detection Reagents Brightfield5

Accessories

DUO80102-40ML Duolink In Situ Brightfield Mounting Medium 40ML

DUO82040-5ML Duolink In Situ Mounting Medium with DAPI 5ML

DUO82047-4L Duolink In Situ Wash Buffers for Brightfield 4L

DUO82047-20L Duolink In Situ Wash Buffers for Brightfield 20L

DUO82049-4L Duolink In Situ Wash Buffers for Fluorescence 4L

DUO82049-20L Duolink In Situ Wash Buffers for Fluorescence 20L

DUO82064-1KT Duolink In Situ Microplate Nuclear Stain and Anti-Fade

2ML

DUO82065-1EA Duolink In Situ Microplate Heat Transfer Block 1 Block

Software

DUO90806-1EA Duolink ImageTool

These products are for research use only. 1 reaction is calculated on 40 μL of reagents to cover 1 square cm.1) Orange – PLA signals detected with the same filters as for e.g. Cy3.2) Red – PLA signals detected with the same filters as for e.g. TexasRed.3) Far Red – PLA signals detected with the same filters as for e.g. Cy5, suitable for

confocal microscopy.4) Green – PLA signals detected with the same filters as for e.g. GFP, suitable with

red counterstaining e.g. propidium iodide.5) Brightfield – PLA signals visualized by enzymatic conversion of HRP/NovaRED

substrate using brightfield microscope.

Duolink In Situ Product ListTo perform a Duolink assay a PLA probe PLUS, a PLA probe MINUS and a Detection Reagent are required. Recommended accessories are Wash Buffers and Mounting Medium.

If species X is:

Mouse

Goat

Rabbit

Goat or Rabbit

Mouse or Rabbit

Mouse or Goat

then species Y should be:

PLA probe PLUS anti-species X

PLA probe MINUS anti-species Y

Primary antibody species Y anti-protein B

Primary antibody species X anti-protein A

To generate a PLA signal, use primary antibodies from Mouse, Goat or Rabbit as shown above. Alternatively, build your own PLA probe using Probemaker kits DUO92009 and DUO92010.

5

Prestige Antibodies® Powered by Atlas Antibodies are the Most Highly Characterized in the Industry• More than 700 IHC, IF and Western blot images for each antibody

• Standardized in detailed universal protocols

• Developed by the Human Protein Atlas (HPA) Project

• Access all data via the Human Protein Atlas website

Since 2008, Sigma has partnered with Atlas Antibodies to provide Prestige Antibodies. Each Prestige Antibody, designed and validated by the Human Protein Atlas Project, is accompanied by 700 immunohistochemistry (IHC), immunofluorescence (IF) and Western blot images. The validation process is supported by publicly available data of the Human Protein Atlas, a part of HUPO’s Human Antibody Initiative. To ensure our Prestige Antibody collection has market leading specificity, each antibody is tested by multiple quality assurance steps.

The Human Protein Atlas (proteinatlas.org) displays a collection of more than 13 million images of normal and cancer tissues, cell lines and primary cells. The database delivers visible proof of antibody performance and also provides a knowledge base demonstrating structural and temporal expression of proteins in various cells and tissues.

For more information, visit sigma.com/prestige

Immunofluorescence

Anti-TJP1: (Cat. No. HPA001636) Immunofluorescent staining of human cell line U-2 OS shows positivity in cytoplasm & plasma membrane.

Immunohistochemistry

Anti-MTHFD1: (Cat. No. HPA000704) Immunoperoxidase staining of formalin-fixed, paraffin-embedded human liver tissue shows strong cytoplasmic and/or membranous staining of hepatocytes.

sigma.com/detection 7

Low Background Detection in Life Science Applications using Sunstone® Luminescent UCP NanocrystalsUpconversion Imaging BackgroundWhile fluorescent imaging applications are commonly used in life science research, inherent limitations to fluorescence, especially for cellular applications, have encouraged development of alternative techniques and compounds. Upconversion luminescence using rare-earth doped nanocrystals is increasingly being used in commercial and industrial applications and more recently has been used in life science applications to overcome these limitations found with quantum dots and other normal fluorophores (see Figure 1).

Upconversionnano-particle

emission excitation

Quantum dots

emissionexcitation

468 nm excitation

980 nm excitation

Fluorescenceof mouse body

F1

A

B

Figure 1. Illustration of fundamental difference between quantum dots with normal fluorescence and upconversion nanocrystals.

Normal fluorescence converts higher-energy (shorter wavelength) light to lower-energy (longer wavelength) emitted light. Upconversion luminescence is based on the absorption of two or more low-energy (longer wavelength, typically infrared) photons by a nanocrystal followed by the emission of a single higher-energy (shorter wavelength) photon (see Figure 2).

300 400 500 600 700 800

Infrared

Visible

Ultraviolet

Wavelength (nm)

Ener

gy

UCP

2γ

γ

Figure 2. Simplified principle of up-conversion luminescence. Two or more low-energy photons (longer infrared wavelength) are absorbed by an upconverting phosphor followed by the emission of a single higher-energy photon (shorter visible wavelength).

Sunstone Upconverting Nanocrystals (Sunstone UCP Nanocrystals)Sunstone Nanocrystals from Intelligent Material Solutions® Inc. are a proprietary, novel series of rare earth-doped nanocrystals of small size, high quantum efficiency, and high photoluminescent intensity functionalized for use in industrial and life sciences applications. These patented materials possess unique and inherent atomic states that allow the conversion of various wavelengths of light energy up and down the electromagnetic spectrum.

Eliminate Autofluorescence in Biological Samples Upconversion bioimaging has significantly lower autofluorescence (see Figure 3) and a higher signal to noise ratio when compared to single-photon excitation for fluorescence detection.

Figure 3. Comparison of Sunstone Upconversion Nanocrystals (left) to fluorescein (right) for imaging of prostate serum antigen (PSA) in human prostate cancer immunohistology sample. Notice the absence of tissue autofluorescence in the sample detected using Sunstone Upconversion nanocrystals (left). Figure provided by Intelligent Materials, Inc. (Dr. Sam Niedbala, Lehigh University)

Large Anti-Stokes Shift for Discrete Emission Signals in Multiplex TechniquesA large anti-Stokes shift (up to 500 nm) with upconversion phosphorescence means well-separated and discrete emission peaks from the infrared excitation source. For NIR emitting nanoparticles, the longer wavelength allows deeper penetration of the exciting light into biological tissues. Furthermore, by varying the concentration and ratio of lanthanides, the decay time of Sunstone nanocrystals can be modified from less than a femtosecond to a millisecond or longer, and these temporal properties are reproducible for a specific formulation, ensuring consistency in replicate tests. Standard detectors can easily differentiate these temporal signatures. A combination of Sunstone Nanocrystals can be applied in an assay using a single excitation source to produce different emission signals for multiplex applications.

Small Particle Size for Life Science Applications Sunstone Nanocrystals available through Sigma-Aldrich are 30 nm rods, which are small enough for life science applications without further processing (see Figure 4). Sunstone Nanocrystals have a high degree of structural homogeneity, allowing for finely tuned temporal properties and narrow spectral emission. The nanocrystals are synthesized in the β crystal phase (β-NaYF4), requiring low phonon energy for upconversion1. Sunstone Nanocrystals exhibit excellent size distribution, uniformity in shape, and high monodispersity.

Additional sizes and morphologies of Sunstone Nanocrystals will be made available in the future to support life science research.

Figure 4. Transmission electron microscope ~30-nm images of rod-shaped NaYF4 Sunstone Upconverting Nanocrystals. Sunstone Nanocrystals exhibit excellent size distribution, uniformity in shape, and high monodispersity.

Resistant to Photobleaching and Fading Sunstone Upconverting Nanocrystals do not photobleach and allow permanent excitation with simultaneous signal integration. They can be stored indefinitely without a decrease in light emitting efficiency for repeated irradiation and analysis.

Biocompatible and Non-Toxic Sunstone Nanocrystals are biocompatible and non-toxic, with no cytotoxicity against human osteosarcoma cells2. Upconverting nanocrystals have been used in in vivo experiments with C. elegans3

and mice4 without demonstrating toxicity.

Life Science ApplicationsUpconverting materials have been used in a broad variety of life science applications including:

• Immunohistochemistry2,5-7

• Immunocytochemistry5-7

• Multiplex immunoassays5,6

• Nucleic acid microarrays6,8,9

• In vivo, in situ, and ex situ biomedical imaging2,3,5,10,11

• Flow cytometry8,12

• Enzymatic assays6,8,12

• Fluorescence resonance energy transfer (FRET) bioanalytical assays12

Sunstone Nanocrystals are ideal for life science detection and imaging with broad applicability in many in vitro and in vivo techniques. Sunstone Nanocrystals are extremely robust and can be incorporated into many chemical and biochemical compositions without losing their phosphorescence as long as the medium in which they are dispersed is optically transparent to both the absorbed and emitted radiation. With sharp emission bands and large anti-Stokes shifts, Sunstone Nanocrystals may be used in multiplex techniques allowing identification of multiple emission spectra from a single sample (see Figure 5). The nanocrystals are also suitable for FRET based assay systems.

Figure 5. ( green) CD4+ lymphocytes labeled with CD4 UCP-conjugated antibody. (blue) calcein dye.

9sigma.com/detection

Sunstone Nanocrystals have been used for in vivo and in situ macroscopic lymphatic imaging in mice (see Figure 6). Multiplex analysis using 980 nm excitation was performed by using a combination of green-emitting and red-emitting upconversion particles.

Figure 6. Luminescent in vivo and in situ lymphatic imaging with infrared Sunstone Upconverting nanocrystals obtained with both spectral and single shot imaging. The nanocrystals depicted the draining lymph nodes during in vivo and in situ imaging in mice. Due to the minimal background, single shot luminescence images obtained at 800 nm were comparable to the spectrally unmixed images which were post processed to remove autofluorescence4. (Dr. Hisataka Kobayashi, National Institutes of Health)

Carboxylated Sunstone Nanocrystals may be attached to biomolecules via the carboxyl group after EDC activation. Carboxylated nanocrystals can also be conjugated via electrostatic binding. Carboxylated Sunstone Nanocrystals can be conjugated to a variety of biomolecules including:

• Antibodies

• Enzymes

• Substrates

• Small molecules and inhibitors

Avidin conjugated Sunstone Nanocrystals can be used in common biotin-avidin/streptavidin reporter systems.

Ordering InformationProduct No. Description Functional group λEx (nm) λEm max. (nm)* Crytsal morphology89157 Sunstone Upconverting nanocrystals UCP 804, carboxylated Carboxyl 976 804 10 - 60 nm, rods61334 Sunstone Upconverting nanocrystals UCP 545, carboxylated Carboxyl 976 545 11 - 60 nm, rods66698 Sunstone Upconverting nanocrystals UCP 538, carboxylated Carboxyl 976 538 12 - 60 nm, rods90838 Sunstone Upconverting nanocrystals UCP 475, carboxylated Carboxyl 976 475 13 - 60 nm, rods76736 Sunstone Upconverting nanocrystals UCP 804, avidin conjugated Avidin 976 804 14 - 60 nm, rods90992 Sunstone Upconverting nanocrystals UCP 545, avidin conjugated Avidin 976 545 15 - 60 nm, rods75207 Sunstone Upconverting nanocrystals UCP 538, avidin conjugated Avidin 976 538 16 - 60 nm, rods89043 Sunstone Upconverting nanocrystrals UCP 475, avidin conjugated Avidin 976 475 17 - 60 nm, rods67345 Sunstone Upconverting nanocrystals UCP 475 none 976 475 10 - 60 nm, rods57688 Sunstone Upconverting nanocrystals UCP 475, amin conjugated amine 976 475 10 - 60 nm, rods74932 Sunstone Upconverting nanocrystals UCP 538 none 976 538 10 - 60 nm, rods52498 Sunstone Upconverting nanocrystals UCP 538, amine conjugated amine 976 538 10 - 60 nm, rods73909 Sunstone Upconverting nanocrystals UCP 538, biotin conjugated biotin 976 538 10 - 60 nm, rods74344 Sunstone Upconverting nanocrystals UCP 545, amine conjugated amine 976 545 10 - 60 nm, rods79882 Sunstone Upconverting nanocrystals UCP 545, biotin conjugated biotin 976 545 10 - 60 nm, rods53367 Sunstone Upconverting nanocrystals UCP 804 none 976 804 10 - 60 nm, rods73702 Sunstone Upconverting nanocrystals UCP 804, amine conjugated amine 976 804 10- 60 nm, rods43177 Sunstone Upconverting nanocrystals UCP 804, biotin conjugated biotin 976 804 10- 60 nm, rods42923 Sunstone Upconverting nanocrystals UCP 475 none 976 545 10 - 60 nm59956 Sunstone Upconverting nanocrystals UCP 475, biotin conjugate biotin 976 475 10 - 60 nm

*Refers to strongest signal.

Sunstone is a registered trademark of Intelligent Material® Solutions Inc. Sunstone upconverting nanocrystals are produced under license from SRI International.

References1. Ye, X., Collins, J.E., Kang, Y., Chen, J., Chen, D.T., Yodh, A.G., and Murray, C.B. Morphologi-cally controlled synthesis of colloidal upconversion nanophosphors and their shape-directed self-assembly. Proc. Natl. Acad. Sci. USA, 107, 22430-5 (2010).2. Shan, J., Chen, J., Meng, J., Collins, J., Soboyejo, W., Friedberg, J.S., and Ju, Y. Biofunction-alization, cytotoxicity, and cell uptake of lanthanide doped hydrophobically ligated NaYF4 upconversion nanophosphors. J. Appl. Phys., 104, 094308 (2008).3. Lim, S.F., Riehn, R., Ryu, W.S., Khanarian, N., Tung, C.K., Tank, D., and Austin, R.H. In vivo and scanning electron microscopy imaging of upconverting nanophosphors in Cae-norhabditis elegans. Nano Lett., 6, 169-74 (2006).4. Kobayashi, H., Kosaka, N., Ogawa, M., Morgan, N., Smith, P.D., Murray, C.B., Ye, X., Collins, J., Kumar, G.A., Bell, H., and Choyke, P.L. In vivo multiple color lymphatic imaging using upconverting nanocrystals. J. Mater. Chem., 19, 6481–84 (2009).5. Bünzli, J-C.G. Lanthanide luminescence for biomedical analyses and imaging. Chem. Rev., 110, 2729–55 (2010).6. Corstjens, P., Chen, Z., Zuiderwijk, M., Bau, H.H., Abrams, W.R., Malamud, D., Sam Nied-bala, R., and Tanke, H.J. Rapid assay format for multiplex detection of humoral immune responses to infectious disease pathogens (HIV, HCV, and TB). Ann. N.Y. Acad. Sci., 1098, 437-445 (2007).7. Wang, M., Mi, C.-C., Wang, W.-X., Liu, C.-H., Wu, Y.-F., Xu, Z.-R., Mao, C.-B., and Xu, S.-K. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4:Yb,Er upconversion nanoparticles. J. Phys. Chem., 113, 19021-7 (2009).8. Abrams, W.R., Barber, C.G., McCann, K., Tong, G., Chen, Z., Mauk, M.G., Wang, J., Volkov, A., Bourdelle, P., Corstjens, P.L., Zuiderwijk, M., Kardos, K., Li, S., Tanke, H.J., Sam Niedbala, R., Malamud, D., and Bau, H. Development of a microfluidic device for detection of patho-gens in oral samples using upconverting phosphor technology (UPT). Ann. N.Y. Acad. Sci., 1098, 375-388 (2007).9. Corstjens, P.L.A.M., Li, S., Zuiderwijk, M., Kardos, K., Abrams, W.R., Niedbala, R.S. and Tanke, H.J. Infrared up-converting phosphors for bioassays. IEE Proc. Nanobiotechnol., 152, 64–72 (2005).

10. Xue, X., Wang, F., and Liu, X. Emerging functional nanomaterials for therapeutics. J. Mater. Chem., 21, 13107-13127 (2011).11. Chatterjee, D.K., and Yong, Z. Upconverting nanoparticles as nanotransducers for photodynamic therapy in cancer cells. Nanomedicine (Lond.), 3, 73 -82 (2008).12. Soukka, T., Kuningas, K., Rantanen, T., Haaslahti, V., and Lövgren, T. Photochemical characterization of up-converting inorganic lanthanide phosphors as potential labels. J. Fluoresc., 15, 513-28 (2005).13. Wang, M., Mi, C.-C., Wang, W.-X., Liu, C.-H., Wu, Y.-F., Xu, Z.-R., Mao, C.-B., and Xu, S.-K. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF(4):Yb,Er upconversion nanoparticles. ACS Nano, 3, 1580-6 (2009).14. Kokko, T., Liljenback, T., Peltola, M.T., Kokko, L., and Soukka, T. Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer. Anal. Chem., 80, 9763–8 (2008).15. Soukka, T., Rantanen, T., and Kuningas, K. Photon upconversion in homogeneous fluorescence-based bioanalytical assays. Ann. N.Y. Acad. Sci., 1130, 188–200 (2008).16. Zijlmans, H.J.M.A.A., Bonnet, J., Burton, J., Kardos, K., Vail, T., Niedbala, R.S., and Tanke H.J. Detection of cell and tissue surface antigens using up-converting phosphors: A new reporter technology. Anal. Biochem., 267, 30–36 (1999).

For more information, visit sigma.com/upconversion


CF dyes are a series of highly water-soluble fluorescent dyes spanning the visible and near-infrared (near-IR) spectrum (Figure 1A and 1B) for labeling antibodies, proteins, nucleic acids, and other biomolecules. Developed by scientists using new breakthrough chemistries, the brightness, photostability, and color selection of CF dyes rival or exceed the quality of other commercial dyes as a result of rational dye design.

Sigma® Life Science currently offers 19 CF dyes spanning the visible and near-IR wavelengths, with additional colors in development.

The CF dye product line includes reactive CF dyes, labeling kits, CF-labeled secondary antibodies, and other bioconjugates. This collection further expands Sigma Life Science’s broad range of carefully selected secondary antibodies and conjugates, allowing scientists to achieve greater sensitivity, brighter results, and better photostability in immunoassays.

The CF dye quick reference guide will help you choose the correct dye for your application.

Absorption/Emission Spectra of Goat Anti-Mouse Antibody

CF ™ DyesAchieve greater sensitivity, brighter results, and better photostability with enhanced CF™ dye technology.

Absorption spectra of CF dyes conjugated to goat anti-mouse IgG

0

20

40

60

80

100

120

250 300 350 400 450 500 550 600 650 700 750 800 850 900Wavelength(nm)

Abs

orpt

ion

1:CF3502:CF405S3:CF405M4:CF488A5:CF5436:CF5557:CF5688:CF5949:CF620R10:CF63311:CF640R12:CF64713:CF660C14:CF660R15:CF68016:CF680R17:CF75018:CF77019:CF790

Emission spectra of CF dyes conjugated to goat anti-mouse IgG

0

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40

60

80

100

120

350 400 450 500 550 600 650 700 750 800 850 900Wavelength(nm)

Fluo

resc

ence

1:CF3502:CF405S3:CF405M4:CF488A5:CF5436:CF5557:CF5688:CF5949:CF620R10:CF63311:CF640R12:CF64713:CF66014:CF660R15:CF68016:CF680R17:CF75018:CF77019:CF790

Figure 1A

Figure 2A

Figure 2B

Figure 2C

Figure 1B

Brightness, Photostability and Conjugate Specificity of CF Dyes Compared to Other Commercial Dyes

Relative fluorescence of CF543 and Alexa Fluor 546 (AF546) goat anti-mouse conjugates as a function of the number of dye molecules per protein (degree of labeling).

Near-IR CF dyes are highly water soluble without carrying excessive negative charge, which can increase non-specific binding of antibody conjugates. Near-IR Western blots imaged using the Odyssey system (Li-COR Biosciences). Two dilutions of HeLa cell lysate were probed with mouse anti-tubulin antibody followed by goat anti-mouse conjugated to Alexa Fluor 790 (AF790) (2, 3) or CF790 (5, 6).

Relative photostability of CF633 and Alexa Fluor 647 (AF647) goat anti-mouse conjugates. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 followed by CF633 or Alexa Fluor 647 goat anti-rabbit IgG conjugates. Cells were imaged using a mercury arc lamp microscope equipped with a Cy5 filter set and CCD camera. Sequential images were captured at 0, 1, and 5 minutes.

For more information, visit sigma.com/cfdyes

Mixing antibody and dye in buffer

Dye conjugated antibody

Label 5 to 100 μg of an Antibody with the Brightest Dyes Available in Just 30 MinutesMix-n-Stain™ CF™ dye antibody labeling kits dramatically simplify he process of preparing fluorescently labeled antibodies, particularly primary antibodies. Simply mix your antibody with the CF dye of your choice in the buffer provided, a step that takes less than 30 seconds of hands-on time. After a quick 30-minute incubation, you will have a ready-to-use fluorescent antibody conjugate.

Advantages for Using Mix-n-Stain™ Labeling Kits• No need to calculate how much dye you should use - just mix your

antibody with the entire amount of dye provided

• No purification of conjugate necessary

• The labeling reaction can tolerate the presence of common stabilizers, such as sodium azide, Tris, and low levels of glycerol, BSA or gelatin.

• Choose from more than 20 of the brightest and most photostable dyes commercially available.

Simple Antibody Labeling Protocol – 3 Easy Steps1. Mix antibody and dye in buffer – 30 seconds hands-on time

2. Incubate antibody/dye for 30 minutes

3. Conjugate is ready-to-use — no purification necessary!

For more information, visit sigma.com/mixnstain

Mix-n-Stain™ Antibody Labeling Kit


Fluorescent Dye Comparison

CF™ dye Reactive formsλEx

(nm)λEm

(nm) MW εExcitation

sourceDirect replacement for: Reactive forms

λEx (nm)

λEm (nm) MW ε Advantages of CF™ dye

CF™ 350amine, aminooxy, hydrazide, maleimide, SE

347 448 496 18,000 UV

AMCA NHS, sulfo-NHS, hydrazide, HPDP, SE 353 442 330 19,000 - Yields the brightest blue fluorescent antibody conjugates when excited at ~350 nm - Highly water-soluble and insensitive to pH

Cascade Blue® Acetyl azide, hydrazid, ethylenediame 400 420 596 28,000

DyLight™ 350 NHS, maleimide 353 432 899 15,000

CF™ 405Samine, aminooxy, maleimide, SE

404 431 1169 33,000 405 nm laserAlexa Fluor® 405 SE, cadaverine 400 424 1028 35,000

- Much brighter than Alexa Fluor® 405 due to better compatibility with excitation and emission windows on common instruments

DyLight™ 405 NHS, maleimide 400 420 793 30,000 - Highly water-soluble and insensitive to pH

CF™ 405Maminooxy, maleimide, SE

408 452 503 41,000 405 nm laser

BD Horizon™ V450

Conjugated 404 448 - More photostable than Pacific Blue® dye - As bright as Pacific Blue® dye in the blue channel - Less spill-over fluorescence in the green channel - Highly water-soluble

eFluor® 450 Conjugated 405 450

Pacific Blue® SE 405 455 406 46,000

CF™ 488Aamine, aminooxy, hydrazide, maleimide, SE

490 515 914 70,000 488 nm laser

Alexa Fluor® 4885-SDPE, 5-TFPE, maleimide, alkyne, azide, cadaverine, hydrazide, hydroxylamine, SE

494 517 643 73,000 - Minimal charge compared to Alexa Fluor® 488 reduces nonspecific antibody staining - Less spill-over fluorescence in the red channel than Alexa Fluor® 488 - Extremely photostable - Highly water-soluble and pH-insensitive

Cy® 2 Bis NHS 489 506 714 150,000

DY-495NHS, maleimide, carboxylic acid, amino derivative

493 521 623 70,000


FAM™ SE 495 520 83,000

FITC 494 518 389 77,000

Fluorescein NHS, maleimide 490 514 332 93,000

CF™ 543amine, hydrazide, maleimide, SE

541 560 870 100,000532 nm or 543, 546

laserAlexa Fluor® 546 N/A 556 573 N/A 112,000

- Significantly brigher than Alexa Fluor® 546- Highly water-soluble and pH-insensitive

CF™ 555amine, hydrazide, maleimide, SE

555 565 810 150,000532 nm or

568 nm laser

Alexa Fluor® 555maleimide, alkyne, azide, cadaverine, hydrazide, SE

555 572 1250 155,000 - Brighter and more photostable than Cy 3 - Minimal charge compared to Alexa Fluor® 555 reduces nonspecific antibody staining

Cy3™ Bis NHS, hydrazide, maleimide, NHS 552 566 767 150,000


557 574 739 150,000


TRITC 557 576 479 100,000


562 583 714 100,000532 nm or

568 nm laser

Alexa Fluor® 568 maleimide, cadaverine, hydrazide, SE 578 602 792 88,000 - Optimal for the 568 nm line of the Ar-Kr mixed-gas laser - Brighter and more photostable than Alexa Fluor® 568


559 578 769 120,000

Rhodamine Red™ NHS 570 590 654 129,000


593 614 729 115,000532 nm or

594 nm laser


590 617 820 92,000 - Yields the brightest antibody conjugates among spectrally similar dyes - Extremely photostableDY-594 NHS, maleimide 589 614 92,000


Texas Red® SE 595 615 702 116,000

CF™ 620R maleimide, SE 617 639 738 115,000633 nm or

635 nm laserLightCycler® Red 640

NHS 625 640 758

- Excellent energy acceptor for FRET - Highly fluorescent - Extremely photostable - Highly water-soluble

CF™ 633amine, aminooxy,

hydrazide, maleimide, SE

630 650 821 100,000633 nm or

635 nm laser

Alexa Fluor® 633 maleimide, hydrazide, SE 621 639 1200 159,000 - Yields the brightest antibody conjugates among spectrally similar dyes Optimal for 633 nm He-Ne laser or the 635 nm red diode laser - Far more photostable than Alexa Fluor® 647 - Highly water-soluble


636 657 732 200,000


CF™ 640Ramine, aminooxy, hydrazide, maleimide, SE

642 662 832 105,000633 nm,

635 nm, or 640 nm laser


651 672 1250 270,000 - Most photostable among spectrally similar dyes - Yields highly fluorescent protein conjugates - Very water-soluble and pH-insensitive

Cy® 5 Bis NHS, hydrazide, maleimide, NHS 649 666 792 250,000

CF™ dye Reactive formsλEx

(nm)λEm

(nm) MW εExcitation

sourceDirect replacement for: Reactive forms

λEx (nm)

λEm (nm) MW ε Advantages of CF™ dye

CF™ 647hydrazide, maleimide, SE

650 665 836 240,000633 nm,



651 672 1250 270,000 - Antibody conjugates have the best signal-to-noise ratio compared to Alexa Fluor® 647, DyLight™ 649 and Cy 5 - Highly fluorescent - Highly water-soluble and pH-insensitive

Cy® 5 Bis NHS, hydrazide, maleimide, NHS 649 666 792 250,000

DY- 647NHS, maleimide, carboxylic acid, amino derivative

653 672 762 250,000


CF™ 660C maleimide, SE 667 685 3112 200,000633 nm,


Alexa Fluor® 660 maleimide, SE 668 698 1100 132,000 - Much brighter than Alexa Fluor® 660 - More photostable than Alexa Fluor® 660 - Highly water-soluble

CF™ 660Raminooxy, maleimide, SE

663 682 888 100,000633 nm,


Alexa Fluor® 660 maleimide, SE 668 698 1100 132,000

- Brighter than Alexa Fluor® 660 - The most photostable 660 nm dye, ideal for confocal microscopy - Highly water-soluble

CF™ 680 maleimide, SE 681 698 3241 210,000680 nm or

685 nm laser

Alexa Fluor® 680 maleimide, SE 684 707 1150 183,000 - The brightest among spectrally similar dyes - Superior signal-to-noise ratio in immunostaining - Highly water-soluble and pH-insensitive

Cy® 5.5 Bis NHS, hydrazide, maleimide, NHS 675 695 1128 250,000


IRDye® 680 NHS, maleimide 676 693 1402 250,000

CF™ 680Raminooxy, maleimide, SE

680 701 912 140,000680 nm or

685 nm laser

Alexa Fluor® 680 maleimide, SE 684 707 1150 183,000 - The most photostable 680 nm dye - Suitable for labeling nucleic acids and small bio-molecules - Highly water-soluble and pH-insensitive - CF680/CF770 double labeling compatible with Li-COR Odyssey System

Cy® 5.5 Bis NHS, hydrazide, maleimide, NHS 675 695 1128 250,000


IRDye® 680 NHS, maleimide 676 693 1402 250,000

CF™ 750 SE 755 777 3009 250,000680 nm,


Alexa Fluor® 750 maleimide, SE 753 782 1300 290,000 - Exceptionally bright and stable - Less immunogenic than competing dyes - Better signal-to-noise ratio compared to APC- Alexa Fluor® 750 tandem dye with 633 nm excitation

Cy® 7 Bis NHS, NHS 743 767 818 250,000


APC- Alexa Fluor® 750

Conjugated 650 779 240,000

CF™ 770 SE 770 797 3138 220,000 785 nm laser

DyLight™ 800 NHS, maleimide 777 794 899 270,000 - Exceptionally bright and stable- Less immunogenic than competing dyes- CF680/CF770 double labeling compatible with Li-COR Odyssey System

IRDye® 800CW NHS, maleimide, carboxylate 778 794 1166 300,000

CF™ 790 SE 784 806 3267 210,000 785 nm laser Alexa Fluor® 790 SE 785 810 1750 260,000- Exceptionally bright and stable- Less immunogenic than competing dyes

Fluorescent Dye Comparison cont’d


Atto™ Dyes for Superior Fluorescent Imaging

Activated fluorescent dyes are routinely used to tag proteins, nucleic acids, and other biomolecules for use in life science applications including fluorescence microscopy, flow cytometry, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET) techniques, receptor binding assays, and enzyme assays. The Atto dyes are a series of fluorescent dyes that meet the critical needs of modern fluorescent technologies:

• Enhanced photostability and ozone resistance

• Long signal lifetimes

• Reduced background for greater sensitivity

• Extensive selection of alternatives to common dyes

• Recommended for multiplex applications

Longer Excitation Wavelengths for Reduced Background Diode laser excitation at 635 nm and red-absorbing fluorescent dyes were shown to reduce autofluorescence of biological samples sufficiently so that individual antigen and antibody molecules could be detected in human serum samples.1,2 Excitation in the red spectral region also reduces cell damage when working with live cells.3

• Many Atto dyes (Atto 590 and above) can be excited using wavelengths greater than 600 nm.

• Using long-wavelength activated Atto dyes with the appropriate excitation wavelength reduces autofluorescence due to sample, solvent, glass, or polymer support.

• Background due to Rayleigh and Raman scattering can be dramatically reduced.

• Improved overall sensitivity in biological analysis and imaging techniques can be obtained since Atto dyes have less interference from fluorophores with shorter lifetimes.

• Fluorescent signals are stronger with the same molar amount of Atto 655 or Atto 680 since less dye is lost to inactivation. Other Atto dyes also have low triplet formation.

ATTO 655

Cy 5

0

1.0

0.8

0.6

0.4

0.2

0.010 20 30 40 50 60

Time (minutes)

Abs

orba

nce

Long Signal LifetimesAtto dyes exhibit longer fluorescence signal lifetimes (0.6-3.8 ns) than either carbocyanine dyes or most of the autofluorescence inherent in cells and biomolecules.

Figure 1: Photostability of Atto 655 compared with Cy® 5. Excitation by focused light of a 250 W halogen lamp.

Fluorescence microscopy of paraffin fixed tissue of oesophagus of rat with staining the carbohydrate subunits of the glycoprotein laminin. The target carbohydrate subunit chitotriose [(GlcNAc)3] is specifically bound to lectin from Phytolacca americana-Atto 488 (Cat. No. 39905, green). Concanavalin A-Atto 564 (Cat. No. 69535, red) is specifically bound to α-D-mannose and α−D-galactose. Nucleus counterstained with DAPI (blue).

Atto 655 and Atto 647N – Photostable and Ozone Resistant for Microarray ApplicationsIn contrast to fluorescein and cyanine-based dyes, Atto 655 and Atto 647N have a more rigid molecular structure and are more photostable under prolonged light exposure (see Figure 1). Excitation and emission wavelengths and the emission signal decay time are relatively insensitive to pH and other environmental changes such as temperature and atmospheric humidity.

Imag

e by

J. Z

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en, I

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witz

erla

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Atto™ Dyes - Superior Tool for Super-Resolution Microscopy ApplicationRecent developments in microscopy application, like e.g. STED microscopy enables resolutions down to 10 nm. These applications require fluorescent dyes, that fulfill superior photo-physical criteria. Some Atto-dyes. Atto 488, Atto 647N, and Atto 655, have proven a suitable for techniques such as PALM, dSTORM, STED, etc..

Atto 655 and Atto 680 — Less Molecular Inactivation for Greater SignalNon-fluorescent triplet states and cis-conformations result in fluctuations that interfere with fluorescent signal yield.4 Dyes such as Cy®5, Cy5.5, or Alexa Fluor® 647 may form cis-isomers and triplet states, which precludes their usage in demanding techniques including fluorescence correlation spectroscopy (FCS), single molecule detection (SMD), and as acceptors in fluorescence resonance energy transfer (FRET).5 Atto 655 and Atto 680 have

low intersystem crossing and lack an isomeric bond so they cannot undergo configuration isomerization.

Recommended for Fluorescent Multiplex DetectionAtto dyes can be used to conjugate probes and biomolecules for multiplex applications. Selection of two Atto dyes with separated emission signals supports multiple excitation and measurement results from a single experiment.

Alternatives to Common FluorophoresWith the extensive selection available, Atto dyes can replace commonly used fluorescent dyes. There are Atto dyes suitable for use with any common excitation light source.

Immunoblot detection of Protein 1 and Protein 2 using two primary antibodies and two anti-IgG-Atto dye conjugates .Imaging was done sequentially using a FLA-3000 Fuji® laser scanner, first at an excitation wavelength of 532 nm with a 580 nm emission filter to detect Atto 550, then at an excitation wavelength of 633 nm with a 675 nm emission filter to detect Atto 633. The image overlay was done using a software tool.

Confocal microscopy (CLSM) image of paraffin fixed tissue of rat stomach. Actin stained with mouse anti–smooth muscle α-actin antibody and Atto 488 anti-mouse IgG (Cat. No. 62197, green), cytokeratin stained with polyclonal rabbit anti-cytokeratin and Atto 647N anti-rabbit IgG (Cat. No. 40839, red).

Fluorophore Recommended Atto dye alternativeAlexa Fluor® 488

Atto 488 FITC

FAM™

JOE™Atto 520

TET™

Alexa Fluor 532 Atto 532

HEX™ Atto 532, Atto Rho6G

TAMRA™ Atto 550

Cy®3 Atto 550

Cy3.5 Atto 565

ROX™ Atto 565, Atto Rho11

Alexa Fluor 594 Atto 590 , Atto 594

Texas Red® Atto 590

Alexa Fluor 633 Atto 633, Atto Rho14

Cy5 Atto 647, Atto 647N , Atto 655

Alexa Fluor 647 Atto 647, Atto 647N, Atto 655

Cy5.5 Atto 680, Atto 700

Light source Main lines (nm) Recommended Atto dyesMercury arc lamp 365, 405, 436, 546 Atto 390, Atto 425, Atto

465, Atto 550, Atto 565

Mercury arc lamp 577 Atto 590, Atto Rho101, Atto 594; Atto Rho13, Atto 610, Atto 611x

Xenon arc lamp Continuum and peaks >800 nm

Atto 610, Atto 620, Atto 647, Atto 647N, Atto 655, Atto 680

Halogen lamp Little UV and violet emission; Higher intensity toward longer wavelengths

Atto 610, Atto 620, Atto 647, Atto 647N, Atto 655, Atto 680

Argon ion laser 488, 514 Atto 488, Atto 520, Atto 532, Atto 550

Argon-krypton laser 488, 514, 647, 676 Atto 520, Atto 647, Atto 647N, Atto 655, Atto 680

Krypton laser 647,676 Atto 647, Atto 647N , Atto 655, Atto Oxa12, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740

He-Ne laser 633 Atto Rho14, Atto 633, Atto 647, Atto 647N

Nd-NAG laser 532 Atto 532, Atto Rho6G, Atto 550, Atto 565, Atto Rho11, Atto Rho 12

Common diode laser 635, 650, 670 Atto 633, Atto 647, Atto 647N, Atto 655, Atto 680


Reactive Atto DyesAtto dyes produce intense fluorescent signals due to strong absorbance and high quantum yields.

• Strong signal intensity — Most Atto dyes have εmax values >100,000.

• Ideal for multiplex techniques using visible and near-IR emission wavelengths — Low excitation/emission overlap and good Stokes’ shift separation.

• Selection and versatility — There is an Atto dye suitable to use with any common excitation light source.

Atto dyes are available as:

• Free acid dyes for all routine staining applications.

• NHS-esters for use in common conjugation protocols.

• Maleimides for use in coupling to thiol-containing groups such as cysteine residues and thiol (-SH) tags added during automated synthesis.

Atto 655, Atto 680, and Atto 700 are quenched by guanosine, tryptophan and related compounds through direct contact between the dye and the quenching agent and using an electron transfer process. Fluorescent quenching of dyes by tryptophan residues in proteins has been used to differentiate unbound (non-fluorescent) protein from protein-antibody (fluorescent) interactions.1

Atto Dyeλabs

[nm]ε max

[m-1 cm-1]λem

[nm]ηem [%]

τem [ns]

Catalog NumberFree Acid NHS Ester Maleimide

Atto 390 390 24,000 479 90 3.8 89313 89204 89740

Atto 425 436 45,000 484 90 3.5 n/a 16805 49349

Atto 465 453 75,000 508 55 2.2 50712 53404 55607

Atto 488 501 90,000 523 80 3.2 41051 41698 28562

Atto 495 495 80,000 527 45 2.4 16951 00379 41022

Atto 520 516 110,000 538 90 3.8 70706 77810 16590

Atto 532 532 115,000 553 90 3.8 06699 88793 68499

Atto 550 554 120,000 576 80 3.2 42424 92835 30730

Atto 565 563 120,000 592 90 3.4 75784 72464 18507

Atto 590 594 120,000 624 80 3.7 70425 79636 39887

Atto 594 601 120,000 627 85 3.5 08637 08741 08717

Atto 610 615 150,000 634 70 3.3 78493 93259 41061

Atto 611X 611 100,000 681 35 2.5 40049 18708 n/a

Atto 620 619 120,000 643 50 2.9 92716 67351 n/a

Atto 633 629 130,000 657 64 3.2 18620 01464 n/a

Atto 647 645 120,000 669 20 2.3 97875 07376 41784

Atto 647N 644 150,000 669 65 3.4 04507 18373 05316

Atto 655 663 125,000 684 30 1.9 93711 76245 80661

Atto 665 663 160,000 684 60 16851 04022 01407

Atto 680 680 125,000 700 30 1.8 94875 75999 04971

Atto 700 700 120,000 719 25 1.5 30674 16986 50611

Atto 725 729 120,000 752 10 0.5 47156 93725 n/a

Atto 740 740 120,000 764 10 0.6 91394 59808 n/a

Fluorescent Signal Information for Atto Dyes

λabs - longest-wavelength absorption maximum εmax - molar extinction coefficient at the longest-wavelength absorption maximumλem - fluorescence maximum ηem - fluorescence quantum yield τem - fluorescence decay time

Convenient Atto™ Dye ConjugatesAn extensive selection of Atto dye conjugates and kits are available, including:

• Protein Labeling Kits

- Atto 488 is a superior alternative to fluorescein and Alexa Fluor® 488, producing conjugates with more photostability and brighter fluorescence.

- Atto 550 is an alternative to rhodamine dyes, Cy®3, and Alexa Fluor 550, offering more intense brightness and increased photostability.

- Atto 594 is an alternative to Alexa Fluor 594 and Texas Red®.

- Atto 647N, an extraordinary highly fluorescent dye, and Atto 655 are alternatives to Cy5 and Alexa Fluor 647.

- Atto 633 is an alternative to Alexa Fluor 633.

• Lectins for carbohydrate binding studies.

• Primary and secondary antibodies for direct and indirect ELISA, immunoblotting, immunohistochemistry, and other protein identification applications.

• Biotin and Streptavidin for avidin/streptavidin/biotin conjugation in applications including ELISA, immunohistochemistry, in situ hybridization, and flow cytometry.

• NTA Nickel conjugates for direct detection of polyhistidine-tagged recombinant proteins.

For more information and a comprehensive list of products, visit sigma.com/atto

References1. Neuweiler, H. et al., Detection of individual p53-autoantibodies by using quenched peptide-based

molecular probes. Angew. Chemie, 41, 4769-73 (2002).

2. Sauer, M. et al., Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers. Appl. Phys. B, 65, 427-31 (1997).

3. Terasaki, M., and Dailey, M. E. , Confocal microscopy on living cells. In Handbook of biological confocal microscopy. Pawley, J. B., Ed. 2nd ed., pp 327-346, Plenum Press, New York (1995).

4. Widengren, J., and Schwille, P., Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy. J. Phys. Chem. A, 104, 6416-28 (2000).

5. Widengren, J. et.al., Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: theory and experimental realizations. J. Phys. Chem. A, 105, 6851-66 (2001).

6. Buschmann, V., Weston, K.D., and Sauer, M., Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Bioconjugate Chem., 14, 195-204 (2003).

Fluorescent microscopy of human skin tissue section (paraffin fixation) with fungal infection. The target carbohydrate chitotriose of the pathogenic fungi are specifically bound to lectin from Phytolacca americana Atto 488 conjugate (Cat. No. 39905) (green). The nuclei are counterstained with DAPI (blue). Image by J. Zbären, Inselspital, Bern.

His-tagged p38 MAPK protein (500 ng – 25 ng) was separated on a 4-20% Tris-glycine SDS-PAGE gel. After fixing and washing, the gel was incubated with Ni-NTA-Atto 647N (Cat. No. 02175) (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 for Ni-NTA-Atto 647N (λex 647 nm, λem 669 nm). The 50 ng band of His-tagged p38-MAPK is observed using fluorescence imaging.


Microscopic methods in life sciences are of tremendous importance for visualization cellular and tissue structures.

In recent years, development has reached a revolution in order to overcome the resolution barrier given by the diffraction limit. New microscopy concepts that enable resolution limits down to about 10 nm and the visualization of cellular structures and molecular interactions reveal new understanding in biological processes.

These super-resolution microscopy principles are based on several technological approaches. Conventional light microscopy enables a resolution limit of about 250 nm in the x- and y- direction and 450 – 700 nm in the z –direction. Super-resolution techniques have overcome the resolution-limit (Point-spread function), by at least a factor of 2. The resolution of super-resolution microscopy depends on the number of points that can be resolved on the structure of interest. Crucial for successful super-resolution imaging is the choice of fluorescent probe. Brightness and high contrast ratio between the states are of great importance. In most super-resolution methods, the states of the probe must be controllable, reversible or irreversible, switchable between a light or a dark state. Depending on the super-resolution method, further photo physical criteria the probe must be fulfilled. Established techniques are:

• STED (Stimulated emission depletion)

• GSDIM (Ground State Depletion)

• PALM (Photoactivated localization microscopy)

• STORM (Stochastic optical reconstruction microscopy)

• RESOLFT [Reversible saturable optical (flurorescence) transitions]

Sigma now offers the superior series of Abberior dyes that are especially designed and tested for super-resolution microscopy such as STED, RESOLFT, PALM, STORM, GSDIM and others. Abberior STAR, Abberior CAGE, Abberior FLIP and Abberior RSFP – the specific requirements of the super-resolution techniques are served with dedicated dye series.

Super-resolution microscopy depends on fluorescent labels more than any other fluorescence imaging technique. Manufactured by Abberior, the STAR, CAGE and FLIP dyes as well as RSFPs are exceptionally bright and photostable and provide optimized photoswitching for RESOLFT and PALM/STORM imaging. They are the only commercially available dyes tailored specifically to the needs of super-resolution microscopy.

Abberior dyes are also exceptionally well suited for confocal microscopy, epifluorescence imaging and single molecule applications. Basically all fluorescence applications which depend on a good signal to noise ratio and low background benefit from the novel Abberior dyes.

Benefits• Optimized for brightness and very low background

• Optimized switching behavior being the key for super-resolution

• All markers are tested for different super-resolution methods

ū Abberior STAR for STED, confocal and epifluorescence imaging

ū Abberior CAGE & FLIP for PALM, STORM and GSDIM

• Abberior dyes are recommended by renowned microscope vendors

• Proprietary, IP protected products

• Detailed characteristics of the dyes provided, e.g. optimal STED wavelength

Superior Super-Resolution Microscopy Application using Abberior® Dyes

Confocal STED

AbberiorSTAR series

Comparison of confocal (left) and STED (right) microscopy application.

Overview of Abberior® dyes

Dyes DescriptionAbsorption Maximum/ λmax

Extinction Coefficient, ε(λ)

Fluorescence Maximum, λfl

Recommended STED

Cat No., NHS activated

Cat No., maleimid activated

Abberior® CAGE 500 for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)

300 nm (caged,pH 7); 501 nm (uncaged, pH 7)

85,000 — 88,000 m-1cm-1 (pH 7, uncaged)

524 nm (pH 7), 523 nm (MeOH)

595-615 nm 44254 92546


304 nm (caged, PBS, pH 7); 518 nm (uncaged, pH 7)

29,000 m-1cm-1

(pH 7, uncaged)541 nm (pH 7) 610 - 640 nm 38977 95705


300 nm (caged,pH 7); 552 nm (uncaged, pH 7)

66,000 m-1cm-1

(pH 7, uncaged)574 nm (pH 7) 650-670 nm 94822 92545

Abberior® FLIP 565 for single-molecule switching microscopy (e.g. PALM, STORM, GSDIM)

314 nm (PBS, pH 7.4) 51,000 m-1cm1

(MeOH)580 nm (PBS, pH 7.4) 79189 92544

Abberior® STAR 440SX for long Stokes STED and 2-color STED application

430 nm (MeOH), 437 nm (PBS, pH 7.4)

30,800 m-1cm1 (MeOH), 22,700 m-1cm1 (PBS, pH 7.4)

501 nm (MeOH), 515 nm (PBS, pH 7.4)

590-620 nm 68221 38361

Abberior® STAR 470SX for long Stokes STED and 2-color STED application

475 nm (MeOH), 477 nm (PBS, pH 7.4)

30,400 m-1cm1 (MeOH), 22,700 m-1cm1 (PBS, pH 7.4)

609 nm (MeOH), 627 nm (PBS, pH 7.4)

740 - 770 nm 95348

Abberior® STAR 488 for STED application 501 nm (PBS, pH 7.4) 86,000 m-1cm1 (MeOH)

524 nm (PBS, pH 7.4) 585 - 605 nm 61048

Abberior® STAR 512 for STED application 517 nm (MeOH), 512 nm (PBS, pH 7.4)

74,000 m-1cm1 (MeOH)

536 nm (MeOH), 530 nm (PBS, pH 7.4)

590 - 620 nm 38922 03004


64,300 m-1cm1 (MeOH)

609 nm (MeOH), 605 nm (PBS, pH 7.4)

690 - 720 nm 38377


63,000 m-1cm1 (MeOH)

659 nm (MeOH), 654 nm (PBS, pH 7.4)

740 - 770 nm 30558 96013

Abberior® STAR 635P for STED application 635 nm (MeOH), 634 nm (PBS, pH 7.4)

80,000 m-1cm1 (water)

655 nm (MeOH), 654 nm (PBS, pH 7.4)

740 - 770 nm no, only Azide derivative available

References1. T. Müller, C. Schumann, A. Kraegeloh, "STED Microscopy and its Applications: New Insights into

Cellular Processes on the Nanoscale", ChemPhysChem 13, 1986–2000 (2012).

2. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP

3. Tim Grotjohann, Ilaria Testa, Marcel Leutenegger, Hannes Bock, Nicolai T. Urban, Flavie Lavoie-Cardinal, Katrin I. Willig, Christian Eggeling, Stefan Jakobs and Stefan W. Hell. Nature 478, 204-208 (13 October 2011)

For a list of Abberior dyes, visitsigma.com/abberior


Spectral Fluorescence Standard KitThe Spectral Fluorescence Standard Kit (Cat. No. 69336) enables a simple characterization of the relative spectral responsivity and the long-term stability of the emission channel of fluorescence instruments under routine measurement conditions and provides the basis for an improved comparability of fluorescence measurement and eventual standardization.

• CD with Improved Software LINKCORRWIN V.1.1.0.0. developed by BAM for the data evaluation. Version1.1.0.0 only performs well in conjunction with BAM-F002a. The CD also contains instructions for use of BAM-F001 – BAM-F005 and LINKCORRWIN.

• Five Spectral Florescence Standards Ready-made from Sigma-Aldrich GmbH, which cover the spectral region of 300 nm to 770 nm as a set.

• Ethanol, absolute (Cat. No. 34923) Addition of aliquots of 10 mL of ethanol to each solid dye yields a solution that can be measured without additional dilution steps.

• BAM Certificate of the normalized corrected emission spectra of BAM-F001 – BAM-F005 (see also CD).

Application of the Emission Correction CurveBottom: Certified normalized corrected emission spectra (solid lines; lc(λem)) of the kit components and uncorrected, i.e. instrument-dependent emission spectra (dashed lines; lu (λem)), measured with the instrument to be calibrated.

Middle: Individual quotient QF00x = lcF00x (λem)/luF00x (λem) for each kit dye equaling 1/s(λ) of the instrument to be calibrated within the spectral region of the respective fluorescence standard.

Top: Combined emission correction curve 1/s(λ) (solid pink line) calculated from the statistically weighted QF00x as well as its reciprocal s(λ) (black solid line).

Corrected emission spectra are obtained by multiplication of measured spectra by the output of LINKCORR, i.e., 1/s(λ).

Determination of the relative spectral responsivity s(λ) of a fluorescence instrument with the Spectral Fluorescence Standard Kit and LINKCORRWIN.

Certified Reference Materials• For the determination of the relative spectral responsivity s (λ) of

fluorescence instruments. 1/s (λ) is termed emission correction curve. Both can be determined by LINKCORRWIN.

• For the determination of corrected, i.e., instrument-independent emission spectra that are comparable across instruments.

• For the characterization of the long-term stability of the emission channel of fluorescence instruments. Fluorescence spectra that are corrected accordingly do not contain contributions from aging of optical components in the emission channel.

Certified PropertiesNormalized corrected emission spectra of BAM-F001 – BAM-F005 in ethanol for T=25 °C. The emission spectra are traceable to the spectral radiance realized and disseminated in Germany by the Physikalisch-Technische Bundesanstalt (PTB).

The Calibration Kit has been tested in an interlaboratory comparison by the National Metrological Institutes NIST (National Institutes of Standards and Technology, USA), NRC (National Research Council, Canada), PTB (Physikalisch-Technische Bundesanstalt, Germany), and BAM (Federal Institute for Material Research and Testing) employing two different dye concentrations and two different measurement geometries, i.e., 0°/90° and 45°/0°.

For more information, please visit sigma.com/spectral

References1. Hollandt, J.; Taubert, R.D.; Seidel, J.; Resch-Genger, U.; Gugg-Helminger, A.; Pfeifer, D.; Monte, C.,

J. Fluoresc. 2005, 15, 311.

2. Resch-Genger, U.; Pfeifer, D.; Pilz, W.; Monte, C.; Hoffmann, A.; Spieles, M.; Rurack, K.; Hollandt, J.; Taubert, D.; Schönenberger, B.; Nording, P., J. Fluoresc. 2005, 15, 325.

3. Resch-Genger, U.; Hoffmann, K.; Nietfeld, W.; Engel, A.; Neukammer, J.; Nietschke, R.; Ebert, B.; Macdonald, R., J. Fluoresc. 2005, 15, 347.

4. Hoffmann, K.; Monte, C.; Pfeifer, D.; Resch-Genger, U., G.I.T. Laboratory Journal 6, 2005, 3, 18.

5. Monte, C.; Pilz, W.; Resch-Genger, U., Proc. SPIE 2005, 5880, 588019-1.

6. Hoffmann, K.; Monte, C.; Pfeifer, D.; Resch-Genger, U., G.I.T. Laboratory Journal 2005, 6, 29.

7. Monte, C.; Resch-Genger, U.; Pfeifer, D.; Taubert, R.D.; Hollandt, J., Metrologia 2009, 16, 589.

8. Monte, C.; Hoffmann, K.; Pfeifer, D.; Hoffmann, A.; Resch-Genger, U., J Fluoresc. 2006, 16, 441.

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