Oligonucleotide-Blocked Streptavidin for Biotinylation Analysis

Binding between streptavidin, or its homologues, to biotin is one of the most widely exploited biological interactions in the biomedical sciences. Controlling the extent of biotinylation is important for meeting the requirements of the intended design and to preserve the native function of the biotin recipient. Within the protein world, a“trial-and-error” optimization approach toward biotinylation reaction conditions is often necessary due to widely varying properties of proteins. Therefore, product analysis is important. We show here that a oligonucleotide-blocked streptavidin, effectively “monovalent streptavidin”, can tag biotin moieties individually and the tagged products visualized via a polyacrylamide gel shift assay to reveal the product distribution, i.e., [protein-(biotin)n] products where n = 1, 2, 3, etc. This is in contrast, and complementary, to current commercially available analytical reagents for biotinylation characterization, which use an absorbance or fluorescence signal to yield the mean number of biotin moieties.