Antibody Biotinylation Protocol

Principle

Biotin-labeled proteins and antibodies have been widely employed in protein analysis, enzyme immunoassays (EIAs), and diagnostics. The biotin molecule has two ring structures: ring I is the imidazolone ring, which is the main site for avidin binding; ring II is a thiophene ring, and the terminal carboxyl group of the valeric acid side chain on C2 is the only structure that binds antibodies and other biological macromolecules. Biotin can be chemically modified to produce a derivative with a range of active groups-activated biotin. Activated biotin can form covalent bonds with the amino groups of biological macromolecules such as proteins and antibodies. Biotin labeling reactions are commonly performed with a biotin N-hydroxysuccinimide (NHS).

Materials

Methods

1. Dilute the protein to be biotinylated to 1 mg/mL with 0.1 mol/L sodium bicarbonate buffer (pH 8.0). The biotinylation volume for general laboratory applications is 1~2.5 mL.

2. Use 0.1 mol/L sodium bicarbonate buffer (pH 8.0) to fully dialyze the protein.

3. Dissolve 1 mg NHSB in 1 mL DMSO and pipette to dissolve.

4. Add 120 μL of NHSB solution (i.e., containing 120 μg of NHSB) to 1 mL of protein solution (i.e., containing 1 mg of protein).

5. Incubate at room temperature for 2 to 4 hours with constant stirring.

6. Add 9.6 μL 1 mol/L NH₄Cl (1 μl for every 25 μg NHSB) and stir at room temperature for 10 minutes.

7. Fully dialyze against PBS at 4℃& to remove free biotin.

8. Place the sample on a 1 mL molecular sieve column, slowly elute with PBS, collect 1 mL/tube, and wash the protein between 1 and 3 mL.

9. Use the A280 or BCA method to determine the protein content of the purified labeled protein.

10. Add sodium azide (final concentration 0.5 g/L) and 1.0 g/L BSA to the sample. Store the combined product at 4℃ in

the dark, or add 50% redistilled glycerin and store at -20℃.

Application

• Western Blotting

Biotin-labeled antibodies can be used in western blotting to detect specific proteins. After electrophoresis and transfer onto a membrane, the biotinylated antibody binds to the target protein, and streptavidin-conjugated enzymes or fluorochromes are used for visualization.

• Immunohistochemistry (IHC)

Biotin-labeled antibodies are widely used in IHC to localize and detect specific antigens or proteins in tissue samples. The biotin-labeled antibodies bind to the target antigen, and then a streptavidin-peroxidase conjugate is applied, which binds to the biotin moiety, allowing visualization of the antigen by enzymatic reactions.

• ELISA and Other Immunoassays

Biotin-labeled antibodies are commonly used in enzyme-linked immunosorbent assays (ELISA) and other immunoassay formats. The biotinylated antibodies serve as detection reagents by binding to the target antigen, and then streptavidin-conjugated enzymes or fluorochromes are added for signal generation.

• Flow Cytometry

Biotin-labeled antibodies are used in flow cytometry to identify and analyze cell surface antigens. The biotinylated antibodies bind specifically to the target antigens on the cell surface, and then fluorochrome-conjugated streptavidin is added to detect the biotin label. This allows for multiparameter analysis of cell populations.

• Magnetic Bead-based Assays

Biotin-labeled antibodies can be used in combination with streptavidin-coated magnetic beads for capture and isolation of specific target molecules. The biotin-labeled antibody binds to the target, and the complex is then captured using streptavidin-coated magnetic beads, allowing for purification or enrichment of the target molecule.

• Studying Protein-Protein Interactions

Biotin-labeled antibodies can be used to study protein-protein interactions. One of the interacting proteins is biotinylated, and the other protein is detected using a streptavidin-conjugated detection system. This technique is commonly used in pull-down assays and proximity-dependent labeling methods.

Case Study

Fig.1 Biotinylated antibody and streptavidin-HRP optimization in ELISA.¹

Reference

1. Lakshmipriya, Thangavel, et al. "Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay." PloS one vol. 11,3 e0151153. 8 Mar. 2016. Distributed under Open Access license CC BY 4.0, with modification. The image was modified by extracting and using only Part (a) of the original image.