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

Sorenson, Alanna E., Askin, Samuel P., and Schaeffer, Patrick M. (2015) In-gel detection of biotin–protein conjugates with a green fluorescent streptavidin probe. Analytical Methods, 7. pp. 2087-2092.

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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.

Item ID: 38347
Item Type: Article (Research - C1)
ISSN: 1759-9679
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© This Open Access Article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

Funders: National and International Research Alliances Program (NIRAP), National Health and Medical Research Council (NHMRC), James Cook University, Queensland Tropical Health Alliance (QTHA)
Date Deposited: 25 Jun 2015 22:59
FoR Codes: 03 CHEMICAL SCIENCES > 0303 Macromolecular and Materials Chemistry > 030302 Nanochemistry and Supramolecular Chemistry @ 50%
03 CHEMICAL SCIENCES > 0301 Analytical Chemistry > 030104 Immunological and Bioassay Methods @ 50%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970103 Expanding Knowledge in the Chemical Sciences @ 100%
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