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Monobromobimane (mBBr), 25 mg (Invitrogen™)

Monobromobimane is essentially nonfluorescent until conjugated, readily reacts with several low molecular weight thiols, including glutathione, N-acetylcysteine, mercaptopurine, peptides and plasma thiols. The glutathione conjugate of monobromobimane has absorption/emission maxima ~394/490 nm.

5-FAM (5-Carboxyfluorescein), single isomer (Invitrogen™)

The single isomer, 5-FAM, contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Fluorescein is the most common fluorescent derivatization reagent for labeling biomolecules. In addition to its relatively high absorptivity, excellent fluorescence quantum yield, and good water solubility, fluorescein has an excitation maximum that closely matches the 488 nm spectral line of the argon-ion laser.

Monochlorobimane (mBCI) (Invitrogen™)

Monochloromobimane is essentially nonfluorescent until conjugated, readily reacts with several low molecular weight thiols, including glutathione, N-acetylcysteine, mercaptopurine, peptides and plasma thiols. The glutathione conjugate of monochlorobimane has absorption/emission maxima ~394/490 nm.

Click-iT™ pHrodo™ iFL Red sDIBO Alkyne for Antibody Labeling (Invitrogen™)

The Click-iT iFL pHrodo Red sDIBO Alkyne for Antibody Labeling is optimized for easy attachment to azido-modified antibodies using copper-free Click chemistry. This sDIBO label can be used with antibodies that have been modified using the SiteClick Antibody Azido Modification Kit or antibodies that have been engineered to contain azido moieties. These sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less "sticky" and give lower signal background in biological samples.

This modular labeling system gives you the option to choose different fluorescent labels for your antibody and attach another molecule via streptavadin or your own molecule via amine-reactive or amine-containing moieties, depending on your assay.

There are multiple Click-iT sDIBO labels to choose from:
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

Learn more about SiteClick labeling technology ›

Custom SiteClick Antibody Labeling Service and sDIBO labels
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.

6-FAM (6-Carboxyfluorescein), single isomer (Invitrogen™)

The single isomer, 6-FAM, contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Fluorescein is the most common fluorescent derivatization reagent for labeling biomolecules. In addition to its relatively high absorptivity, excellent fluorescence quantum yield, and good water solubility, fluorescein has an excitation maximum that closely matches the 488 nm spectral line of the argon-ion laser.

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. In addition to reactive dye formulations, we offer Alexa Fluor® 405 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

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
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

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.

6-IAF (6-Iodoacetamidofluorescein) (Invitrogen™)

The thiol-reactive 6-iodoacetamidofluorescein (6-IAF) can be used to produce bioconjugates with the 5-isomer of fluorescein.

Alexa Fluor™ 750 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 750 is a bright and photostable near-IR dye that is spectrally similar to Cy7. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 750 dye is water soluble and pH-insensitive from pH 4 to pH 10. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. In addition to reactive dye formulations, we offer Alexa Fluor® 750 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The NHS ester (or succinimidyl ester) of Alexa Fluor® 750 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® 750 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 753/782 nm
Extinction coefficient: 290,000 cm-1M-1
Spectrally similar dyes: Cy7
Molecular weight: ~1300

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
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

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.

Monobromobimane (mBBr), FluoroPure™ grade (Invitrogen™)

Because it is manufactured at our ISO 9001—certified facilities in Eugene, Oregon, we can guarantee that FluoroPure grade monobromobimane is greater than or equal to 98% pure by HPLC.

PyMPO, SE, 1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-Methoxyphenyl)Oxazol-2-yl)Pyridinium Bromide (Invitrogen™)

The amine-reactive PyMPO succinimidyl ester can be used to create bioconjugates with this environment-sensitive fluorophore.

IAEDANS (1,5-IAEDANS, 5-((((2-Iodoacetyl)amino)ethyl)amino)Naphthalene-1-Sulfonic Acid) (Invitrogen™)

The fluorescence of the thiol-reactive IAEDANS is quite dependent upon environment. Its conjugates frequently respond to ligand binding by undergoing spectral shifts and changes in fluorescence intensity that are determined by the degree of aqueous solvation. Advantages of this reagent include high water solubility above pH 4 and a relatively long fluorescence lifetime (10-15 nanoseconds), making the conjugates useful for fluorescence polarization and rotational studies. In addition, because it has a large Stokes shift and an emission that overlaps with the absorption of fluorescein, Alexa Fluor® 488, Oregon Green® dyes and BODIPY® FL dyes, IAEDANS is an excellent reagent for fluorescence resonance energy transfer (FRET) measurements.

CMNB-Caged Carboxyfluorescein, SE (5-Carboxyfluorescein-Bis-(5-Carboxymethoxy-2-Nitrobenzyl) Ether, β-Alanine-Carboxamide, Succinimidyl Ester) (Invitrogen™)

Conjugation of the succinimidyl ester of the water-soluble, CMNB-caged carboxyfluorescein, SE to a primary amine on a biomolecule of interest produces an essentially nonfluorescent probe that yields a green-fluorescent fluorescein-labeled product only after ultraviolet illumination and removal of the caging group.

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

Biotin-XX, SSE (6-((6-((Biotinoyl)Amino)Hexanoyl)amino)Hexanoic Acid, Sulfosuccinimidyl Ester, Sodium Salt) (Invitrogen™)

Biotin-XX, SSE reacts efficiently and reproducibly with amines. The biotin is well separated from the point of attachment by a 14-atom spacer consisting of two, seven-atom aminohexanoyl groups (X). This spacer enhances the accessibility of biotin to the relatively deep biotin-binding sites of avidin and streptavidin. The sulfonate group on the succinimidyl ester increases the water solubility of the reactive moiety.

Sulfo-SHPP (Water-Soluble Bolton-Hunter Reagent) (Thermo Scientific™)

Thermo Scientific Pierce Sulfo-SHPP, sulfosuccinimidyl-3 -(4-hydroxypheynyl) propionate, also known as water-soluble Bolton-Hunter Reagent, conjugates tyrosine-like functional groups to primary amines to increase the number of tyrosyl groups that can be iodinated by iodine-125 labeling procedures.

Features of Sulfo-SHPP:

• Iodinate before or after coupling Sulfo-SHPP to the molecule of interest
• Useful for introducing tyrosyl groups on proteins sensitive to small amounts of organic solvent
• Useful for labelling cell surfaces without exposing the cells to membrane permeable solvents