Identification of Insulin-Activated Rab Proteins in Adipose Cells Using Bio-ATB-GTP Photolabeling Technique.

Francoise Koumanov, Geoffrey D Holman

Methods in molecular biology (Clifton, N.J.) 2018

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We have recently developed a photolabeling method to identify GTP-loaded Rab proteins. The new biotinylated GTP analogue (Bio-ATB-GTP) binds to GTP-binding proteins and after a UV irradiation a covalent bond is formed between the protein and the photoreactive diazirine group on the photolabel. The tagged protein can then be isolated and detected using the classic biotin-streptavidin interaction. In this chapter, we describe the Bio-ATB-GTP photolabel and discuss the advantages of using this photolabeling approach to detect GTP-loaded Rab proteins compared to other existing methodologies. We also describe a step-by-step procedure for detecting the activated state of a Rab protein in primary rat adipocytes.

Citation

Francoise Koumanov, Geoffrey D Holman. Identification of Insulin-Activated Rab Proteins in Adipose Cells Using Bio-ATB-GTP Photolabeling Technique. Methods in molecular biology (Clifton, N.J.). 2018;1713:137-150