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- If using biotin-NHS for protein conjugate, do not use Tris buffer, because it contains nitrogen groups and can quench the reaction. PBS or HEPES as a good buffer.
- Dissolve biotin in DMSO at 1 mg / ml before use.
- Add protein or peptide solution at 1/10 dilution, and incubate on ice for 30 minutes or room temperature for 2 hours at pH 7.5-8.5 for biotin-NHS and at a pH of 6.5-7.5 -maleimide biotin or biotin- for BMCC.
- Quench the reaction with Tris to biotin-NHS, and with beta-mercaptoethanol or dithiothreitol to biotin-maleimide or biotin-BMCC.
- The biotin-tagged protein or peptide can be purified on a streptavidin column, or run on a protein gel and blotted with streptavidin-conjugated enzyme.
- Using Bovine Serum Albumin (BSA) as a Blocking Agent
- Generation of an Affinity Column with Sepharose