Interaction of Arl1 GTPase with the GRIP domain of Golgin-245 as assessed by GST (glutathione-S-transferase) pull-down experiments.

Arl1 is a member of the Arf/Arl family of Ras-like GTPase superfamily. Arl1 is enriched in the trans-Golgi network (TGN). We have recently shown that Arl1 regulates TGN recruitment of GRIP domain-containing Golgin-97 and Golgin-245 by interacting with the conserved GRIP domain present in their carboxyl (C)-termini. We describe here methods for the analysis of the interaction between Arl1(GTP) and the GRIP domain of Golgin-245 using in vitro GST pull-down experiments. GST-Arl1(GTP) can recover endogenous Golgin-245 from HeLa cell cytosol. Furthermore, GST-GRIP domain of Golgin-245 can efficiently retain endogenous active Arl1. A pull-down assay is developed to quantify the relative level of active Arl1.