ImmunoProbe^™ Biotinylation Kit

For custom biotinylation of proteins, Sigma offers kits for conjugation on two different scales. The biotin-avidin system has become popular when high sensitivity and specificity are desired. Biotin can be conjugated to antibodies, lectins, enzymes, and other proteins. The protocols in the kits have been optimized for antibodies. Avidin binds to biotin with a high affinity (K_a = 10¹⁵ M) and specificity. When conjugated to enzymes or fluorochromes avidin provides a means of identifying biotinylated compounds via enzymatic conversion of substrate to form a visible product or detection of fluorescence by spectrophotometry or flow cytometry.

ImmunoProbe^™ Biotinylation Kit has been used for preparation of biotin labeled antibodies.

Biotin has been modified with aminocaproate, then activated via an ester linkage with sulfo-N-hydroxysuccinimide (BAC-Sulfo-NHS). Aminocaproate provides a six-carbon spacer that reduces steric hindrance on the biotin and improves accessibility to the binding site on avidin. Sulfonation of the hydroxysuccinimide increases the polarity of the reagent, allowing it to dissolve easily in aqueous buffer. The ester provides a carbonyl carbon adjacent to a labile ester linkage as a target for primary amine side chains of accessible lysine residues, joining the biotinamidocaproate to the protein via an amide bond.

• Complete protocols for labeling and assay
• BAC-Sulfo-NHS is completely water soluble
• No DMF or DMSO needed
• Biotinylation occurs near neutral pH and physiological ionic strength, avoiding harsh conditions that could damage sensitive proteins
• Fast separation of conjugate from reactants using gel filtration
• 2-5 molar ratio biotin to protein in conjugate (for antibodies)

• Two scales to choose from:
1 mg protein per reaction (B-TAG)
10 mg protein per reaction (BK-101)
• Sufficient reagents for at least 5 labelings

Kit includes all reagents necessary for biotinylation and for determination of biotin/protein molar ratio.

Procedure:
Conjugation is performed in four easy steps:
1. Reconstitute BAC-Sulfo-NHS with Phosphate Buffer (PB).
2. Add BAC-Sulfo-NHS to protein and allow to react for 30 minutes at room temperature.
3. Separate the conjugate from the reactants on gel filtration.
4. Assay the conjugate for biotin incorporation by the avidin-HABA assay (BK-101 only). Conjugate is ready to use.

ImmunoProbe is a trademark of Sigma-Aldrich Co. LLC