In-gel detection of biotin–protein conjugates with a green fluorescent streptavidin probe

Abstract

Exploitation of the (strept)avidin-biotin interaction is extremely valuable in a variety of biotechnological applications. Biotin is often covalently linked to proteins or nucleic acids. Determination of the degree of biotinylation of such macromolecules is essential for downstream applications. There is currently a gap in simple yet efficient assays for rapidly quantitating protein biotinylation, as staple methods may produce unclear results or rely on immuno-or competitive assays. We present a simple and reliable electrophoretic method to determine the relative extent of biotinylation of macromolecules. The method relies on complex formation between a biotinylated macromolecule and a streptavidin probe resulting in an electrophoretic mobility shift of the complex detectable by SDS-PAGE. Finally, a green fluorescent protein labelled streptavidin probe was developed to eliminate the need for staining and reduce assay time.

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors

“…S4A) revealing a preferential destabilizing effect of 10a and 10b on Ec BirA. Using a GFP-based biotinylation activity assay, 11,21 we confirmed that 10b leads to Ec BirA-GFP inactivation (Fig. 5B).…”

Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation

“…5A ). Peptide 5 was utilized in a traditional streptavidin-shift assay 35 , since its split-intein mediated capture and protein trans splicing was more effective than with peptides 6 and 7 (Fig. 5B ).…”

“…Taking advantage of the well-documented strong and SDS-resistant binding between streptavidin and biotin, a streptavidin gel-shift assay (adapted from Fairhead and Howarth and Sorensen et al . 35 , 44 ) was used to confirm transfer of biotin moiety to hCNTF; the described PTS reaction setups yielding the putative non-biotinylated and biotinylated hCNTF products were mixed with streptavidin in PBS and the streptavidin-biotin binding allowed 3 hours in room temperature. Detection of hCNTF biotinylation and subsequent streptavidin binding was carried out by Western Blotting.…”

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules

“…Proteins were purified as described for His-tagged proteins. Biotinylation yields were evaluated using either the Pierce Biotin quantitation Kit following the manufacturer's protocol (Thermo Fisher Scientific) or by an SDS-PAGE assay 66 .…”