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Pacific Blue™ Succinimidyl Ester (Invitrogen™)

The amine-reactive Pacific Blue™ succinimidyl ester can be used to can be used to create blue-fluorescent bioconjugates with excitation/emission maxima ~410/455 nm that are excitable by the 405 nm spectral line of the blue diode (violet) laser.

View all Pacific Blue™ dye products..

View the Fluorophore Selection Guide.

CellTrace™ BODIPY™ TR Methyl Ester (Lipophilic Counterstain For GFP) (Invitrogen™)

The red-fluorescent CellTrace™ BODIPY® TR methyl ester is an excellent counterstain for cells and tissues that are expressing GFP. CellTrace™ BODIPY® TR methyl ester readily permeates cell membranes and selectively stains mitochondria and endomembranous organelles such as endoplasmic reticulum and the Golgi apparatus, but does not appear to localize in the plasma membrane. CellTrace™ BODIPY® TR methyl ester staining is retained after paraformaldehyde fixation.

SiteClick™ Antibody Azido Modification Kit (Invitrogen™)

Create a label-ready site-specific azido-modified antibody without lengthy and inefficient genetic modification using the SiteClick Antibody Azido Modification Kit. SiteClick labeling uses enzymes to specifically attach an azido moiety to the heavy chains of an IgG antibody, ensuring that the antigen binding domains remain unaltered for binding to the antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. Once azido–modified, a variety of sDIBO alkyne labels are available for attachment to the antibody via Click chemistry (see list below). This provides the flexibility to choose different labels for your antibody depending on your assay.

Features of the SiteClickAntibody Azido Modification Kit:
• Contains everything required to label 100–250 µg of IgG antibody
• Easy-to-follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate

Learn more about SiteClick labeling technology ›

Related products
Click-iT Alexa Fluor 488 sDIBO Alkyne for Antibody Labeling
Click-iT Alexa Fluor 555 sDIBO Alkyne for Antibody Labeling
Click-iT Alexa Fluor 647 sDIBO Alkyne for Antibody Labeling
Click-iT Biotin sDIBO Alkyne for Antibody Labeling
Click-iT Amine sDIBO Alkyne for Antibody Labeling
Click-iT SDP Ester sDIBO Alkyne for Antibody Labeling

Custom SiteClick Antibody Labeling Service
If you have an antibody that is considered "difficult to label" or has lost activity after labeling using a conventional method, please contact our custom service representatives to determine whether the SiteClick Antibody Labeling Service would be right for your antibody. We offer complete custom SiteClick antibody labeling services with the option of multiple detection molecules including biotin, Alexa Fluor dyes, Qdot fluorophores, R-PE, chelates for PET imaging, and many others.

DyLight™ 775-B2 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight near-infrared specialty dyes, comparable to Alexa Fluor and IRDye NIR dyes, can be used to label antibodies, peptides, and other proteins at primary amines. DyLight 775-B2 dye has a structure based on the benzopyrillium core, with 2 sulfonates. It has excitation and emission peaks at 772 and 787 nm, respectively (in ethanol).

General characteristics of DyLight near-infrared emitting specialty dyes:

Large selection—the largest family of dyes available for NIR fluorescence applications
NHS ester reactive group—allows immediate labeling of antibodies, proteins, peptides and other amine-containing molecules through amide bond formation
Broad spectrum of water solubilities—choose from hydrophilic to hydrophobic dyes to optimize the right dye label for the best performance in a given application
NIR dyes avoid background interference—DyLight NIR Dyes avoid fluorescence interference or quenching effects from biomolecules present in samples
Excellent signal penetration through cells and tissues—DyLight NIR Dyes provide the optimal window for excitation and emission for in vivo imaging applications

DyLight NIR Dyes are a family of labeling agents that can be used for bright fluorescence detection in cell-based imaging or in vivo imaging applications. NIR dyes can be selected based upon their characteristic excitation and emission properties or relative hydrophilicity and hydrophobicity attributes. Dyes that contain a greater number of negatively charged sulfonates generally will have greater water solubility than dyes with fewer sulfonates. More hydrophobic dyes often provide better cell penetrating ability in vivo, while more hydrophilic dyes have less nonspecific binding potential. Each dye contains an amine-reactive NHS ester for simple modification of antibodies, proteins, peptides or other biomolecules through amide bond formation. NIR dyes are best for imaging through tissues and away from indigenous fluorescent biomolecule interference or quenching. DyLight Near Infrared Dyes represent the largest selection of fluorescent labels that are commercially available.

Criteria to consider when choosing a DyLight NIR Specialty Dye
• Excitation and emission wavelengths—choose the best dye to match the excitation and emission capabilities of your instrument
• Water solubility—choose a DyLight NIR Dye based on its relative hydrophilicity, which directly correlates to the number of negatively-charged sulfonates it has on its core structure. More hydrophilic dyes are best at maintaining water solubility of a labeled antibody and limiting the nonspecific binding of the conjugate. More hydrophobic dyes often are best at penetrating tissues and cell membranes in vivo, meaning that dyes with fewer sulfonates may work best for some applications.
• DyLight Dye selection—the broad selection of NIR dyes allows a number of candidate dyes to be tested in a given application for optimal performance.

Applications:
In vivo or ex vivo imaging
• Tumor imaging with labeled peptides
• NIR fluorescence (NIRF) imaging of labeled silica nanoparticles
• NIR in vitro imaging and characterization
• Determination of thermal stability
• Cytotoxicity assays
• Molecular imaging
• UV-VIS-NIR spectroscopy
• Fluorescence correlation spectroscopy
• MRI applications
• DNA sequencing
• Primer labeling for PCR
• 2-D gel electrophoresis
• Flow cytometry/fluorescence-activated cell sorting (FACS)
• Laser scanning confocal microscopy

Related Products
DyLight™ 775-B3 NHS Ester
DyLight™ 775-B4 NHS Ester

TRITC (5/6-tetramethyl-rhodamine isothiocyanate), mixed isomer (Thermo Scientific™)

Thermo Scientific TRITC is a high-performance derivative of rhodamine dye, activated for easy and reliable labeling of antibodies, proteins and other molecules for use as fluorescent probes.

Features of TRITC:

Amine-specific labeling—TRITC varieties of rhodamine efficiently label antibodies and other purified proteins at primary amines (lysine side chains)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescence

Tetramethylrhodamine isothiocyanate (TRITC) is an amine-reactive derivative of rhodamine dye that has wide-ranging application as antibody and other probe labels for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as western blotting and ELISA.

Applications:
• Label antibodies for use as immunofluorescent probes
• Label oligonucleotides for hybridization probes
• Detect proteins in gels and on western blots

Properties of Rhodamine Dyes:
Thermo Scientific Pierce Rhodamine Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

TRITC is the base tetramethylrhodamine molecule functionalized with an isothiocyanate reactive group (—N=C=S) at one of two hydrogen atoms on the bottom ring of the structure. This derivative is reactive towards primary amine groups on proteins, peptides and other biomolecules.

Application Data:

Related Products
Pierce™ NHS-Rhodamine Antibody Labeling Kit
NHS-Rhodamine (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester), mixed isomer

Oregon Green™ 488-X, Succinimidyl Ester, 6-isomer (Invitrogen™)

The amine-reactive Oregon Green® 488-X, succinimidyl ester can be used to can be used to create green fluorescent bioconjugates with excitation/emission maxima ~496/524 nm. This fluorinated analog of fluorescein overcomes some of the key limitations of fluorescein, including greater photostability and a lower pKa (pKa ~ 4.7 versus 6.4 for fluorescein), making its fluorescence essentially pH insensitive in the physiological pH range. This reactive dye contains an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

CellTrace™ Calcein Green, AM - Special Packaging (Invitrogen™)

CellTrace calcein green AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. In live cells the nonfluorescent CellTrace calcein green AM is converted to a green-fluorescent calcein after intracellular esterases remove the acetoxymethyl (AM) esters. This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). For a longer wavelength version of this dye, check out our CellTrace calcein red-orange AM (C-34851).

SiteClick™ Biotin Antibody Labeling Kit (Invitrogen™)

Unlike the conventional amine-thiol crosslinker labeling method, the SiteClick Biotin Antibody Labeling Kit specifically attaches the label to the heavy chains of an IgG antibody, ensuring that the antigen-binding domains remain available for binding to the antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. In addition, no harsh reduction steps are required, and the labeling is consistent and reproducible.

Features of the SiteClickBiotin Antibody Labeling Kit:
• Contains everything required to label 100–250 µg of IgG antibody
• Easy-to-follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate

Learn more about SiteClick labeling technology ›
Learn more about streptavidin detection ›
Learn more about protein and antibody labeling › (We offer a wide selection of antibody and protein labeling kits.)

Custom SiteClick Antibody Labeling Service
If you have an antibody that is considered "difficult to label" or has lost activity after labeling using a conventional method, please contact our custom service representatives to determine whether the SiteClick Antibody Labeling Service would be right for your antibody. We offer complete custom SiteClick antibody labeling services with the option of multiple detection molecules including biotin, Alexa Fluor dyes, Qdot fluorophores, R-PE, chelates for PET imaging, and many others.

CellTracker™ Deep Red Dye (Invitrogen™)

CellTracker™ Deep Red is a fluorescent dye well suited for monitoring cell movement or location. After loading the cells, the dye is well retained, allowing for multigenerational tracking of cellular movements. The deep red excitation/emission spectra are ideal for multiplexing with green and red fluorescent dyes and proteins.

Need a different emission spectrum or longer tracking? View our other mammalian cell tracking products.

• Easy to use—remove medium, add dye, incubate 30 minutes, and image cells
• Fluorescent signal retention of >72 hours (typically three to six generations)
• Red excitation/emission spectra (630/650 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

The CellTracker™ Deep Red fluorescent dye has been designed to freely pass through cell membranes into cells, where it is transformed into cell-impermeant reaction products. CellTracker™ Deep Red dye is retained in living cells through several generations. The dye is transferred to daughter cells but not adjacent cells in a population. CellTracker™ Deep Red dye is designed to display fluorescence for at least 72 hours, and the dye exhibits ideal tracking properties: it is stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. Additionally, the excitation and emission spectra of CellTracker™ Deep Red dye are well separated from GFP (green fluorescent protein) and RFP (red fluorescent protein) spectra allowing for multiplexing.

Alexa Fluor™ 405 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor™ 405 is a blue-fluorescent dye optimal for use with the 405 nm violet laser. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor™ 405 dye is water soluble and pH-insensitive from pH 4 to pH 10.

View all Alexa Fluor® 405 dye products.

View the Fluorophore Selection Guide.

The NHS ester (or succinimidyl ester) of Alexa Fluor™ 405 is the most popular tool for conjugating this dye to a protein or antibody. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor™ conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor™ NHS ester:

Fluorophore label: Alexa Fluor™ 405 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 400/424 nm
Extinction coefficient: 35,000 cm-1M-1
Spectrally similar dyes: Pacific Blue
Molecular weight: 1028.3

Typical conjugation reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide (the provided protocol is optimized for IgG antibodies). You can scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The Alexa Fluor™ NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) (D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate purification
Labeled antibodies are typically separated from free Alexa Fluor™ dye using a gel filtration column, such as Sephadex™ G-25, BioGel™ P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
• Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
• Antibody Conjugate Purification Kit for 20-50 µg (A33087)
• Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn more about protein and antibody labeling
View a selection guide for all Protein Labeling Kits.

View a selection guide for all Antibody Labeling Kits.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

DyLight™ 488-Phosphine (Thermo Scientific™)

Thermo Scientific DyLight Fluor Phosphine Reagents are phosphine-activated fluorescent dyes for specific labeling and detection of azide-tagged molecules, which enables use of fluorescence imaging in metabolic labeling strategies.

When used in combination with azide labeling strategies, phosphine-activated DyLight Fluors enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. The phosphine group conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

General features of the phosphine-activated DyLight Fluors:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents, and does not interfere with fluorescence applications
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that fluorescent labeling is specific
High-performance fluorescence—DyLight 488, 550 and 650 are intense, highly stable fluorophores for green, orange and red fluorescent detection. (see DyLight Fluors)

Related Products
DyLight™ 550-Phosphine
DyLight™ 650-Phosphine

DyLight™ 775-B3 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight near-infrared specialty dyes, comparable to Alexa Fluor and IRDye NIR dyes, can be used to label antibodies, peptides, and other proteins at primary amines. DyLight 775-B3 dye has a structure based on the benzopyrillium core, with 3 sulfonates. It has excitation and emission peaks at 770 and 788 nm, respectively (in ethanol).

General characteristics of DyLight near-infrared emitting specialty dyes:

Large selection—the largest family of dyes available for NIR fluorescence applications
NHS ester reactive group—allows immediate labeling of antibodies, proteins, peptides and other amine-containing molecules through amide bond formation
Broad spectrum of water solubilities—choose from hydrophilic to hydrophobic dyes to optimize the right dye label for the best performance in a given application
NIR dyes avoid background interference—DyLight NIR Dyes avoid fluorescence interference or quenching effects from biomolecules present in samples
Excellent signal penetration through cells and tissues—DyLight NIR Dyes provide the optimal window for excitation and emission for in vivo imaging applications

DyLight NIR Dyes are a family of labeling agents that can be used for bright fluorescence detection in cell-based imaging or in vivo imaging applications. NIR dyes can be selected based upon their characteristic excitation and emission properties or relative hydrophilicity and hydrophobicity attributes. Dyes that contain a greater number of negatively charged sulfonates generally will have greater water solubility than dyes with fewer sulfonates. More hydrophobic dyes often provide better cell penetrating ability in vivo, while more hydrophilic dyes have less nonspecific binding potential. Each dye contains an amine-reactive NHS ester for simple modification of antibodies, proteins, peptides or other biomolecules through amide bond formation. NIR dyes are best for imaging through tissues and away from indigenous fluorescent biomolecule interference or quenching. DyLight Near Infrared Dyes represent the largest selection of fluorescent labels that are commercially available.

Criteria to consider when choosing a DyLight NIR Specialty Dye
• Excitation and emission wavelengths—choose the best dye to match the excitation and emission capabilities of your instrument
• Water solubility—choose a DyLight NIR Dye based on its relative hydrophilicity, which directly correlates to the number of negatively-charged sulfonates it has on its core structure. More hydrophilic dyes are best at maintaining water solubility of a labeled antibody and limiting the nonspecific binding of the conjugate. More hydrophobic dyes often are best at penetrating tissues and cell membranes in vivo, meaning that dyes with fewer sulfonates may work best for some applications.
• DyLight Dye selection—the broad selection of NIR dyes allows a number of candidate dyes to be tested in a given application for optimal performance.

Applications:
In vivo or ex vivo imaging
• Tumor imaging with labeled peptides
• NIR fluorescence (NIRF) imaging of labeled silica nanoparticles
• NIR in vitro imaging and characterization
• Determination of thermal stability
• Cytotoxicity assays
• Molecular imaging
• UV-VIS-NIR spectroscopy
• Fluorescence correlation spectroscopy
• MRI applications
• DNA sequencing
• Primer labeling for PCR
• 2-D gel electrophoresis
• Flow cytometry/fluorescence-activated cell sorting (FACS)
• Laser scanning confocal microscopy

Related Products
DyLight™ 775-B2 NHS Ester
DyLight™ 775-B4 NHS Ester

HABA (4'-hydroxyazobenzene-2-carboxylic acid) (Thermo Scientific™)

Thermo Scientific Pierce HABA is 4'-hydroxyazobenzene-2-carboxylic acid, a simple reagent that enables spectrophotometric (colorimetric) estimation of biotinylation levels of labeled proteins and other molecules.

Features of HABA:

• HABA-avidin complex can be used over a wide range of pH and salt concentrations
• Amount of Avidin can be calculated directly from the increased absorbance at 500nm complexing with the HABA Dye
• Calculate results directly from absorbance values based on extinction coefficients using the procedure outlined in the instructions
• Complete kits also available! See Pierce Biotin Quantitation Kit (Part No. 28005) and Fluorescence Biotin Quantitation Kit (Part No. 46610)

Determine the molar ratio of biotin incorporated into a protein using the HABA-Avidin method. HABA dye (4'-hydroxyazobenzene-2-carboxylic acid ) binds to avidin to produce a yellow-orange colored complex which absorbs at 500nm. Free biotin will displace the HABA dye and cause the absorbance to decrease. A standard curve can be established using the free biotin to estimate the number of moles of biotin incorporated after biotinylating a protein. View online HABA Calculator.

Click-IT™ Biotin Protein Analysis Detection Kit (Invitrogen™)

The Click-iT® Biotin Glycoprotein Detection Kit provides the second part of a simple and robust technique to identify and characterize glycoproteins by Western blot. In step two, after the incorporation of an azide moiety into protein glycan structures with either a Click-iT® metabolic labeling reagent or the Click-iT® Enzymatic Labeling system, the azide-modified glycoproteins are detected via the chemoselective ligation or click reaction between an azide and an alkyne. With this technique, detection sensitivity is in the low femptomole range and biotin-labeled samples can be detected before or after probing the Western with a primary antibody.

Eosin-5-Maleimide (Invitrogen™)

Bioconjugates made with the thiol-reactive eosin-5-maleimide can be used as phosphorescent probes or as photosensitizers. With its high quantum yield (~0.57) for singlet oxygen generation, eosin and its conjugates can be used as effective photooxidizers of diaminobenzidine (DAB) in high-resolution electron microscopy studies and in correlated fluorescence and electron microscopy applications.