FLAG antibody (Agilent Technologies) for Western Blotting

Rab proteins constitute the largest family of monomeric small GTPases that oscillate between active (GTP- bound) and inactive (GDP- bound) conformations. The spatio-temporal activation of Rabs is controlled by the GDP-GTP exchange factors (GEFs), mediating their activation, and the GTP hydrolysis activating proteins (GAPs), terminating their activity. Rab GTPases execute diverse tasks in membrane trafficking by driving cargo collection into nascent transport vesicles, linking vesicles to cytoskeletal elements, recruiting tethers, and finally docking vesicles to the target compartment, leading to membrane fusion. The goal of this project is to characterize a specific GEF for Golgi localized Rab6 GTPase, and to analyze its effect in Rab6-mediated processes at the Golgi complex. In the present experiment, FLAG-tagged Rgp1 was over-expressed in HEK293T cells. FLAG antibody blotting was performed to assess the levels of over expressed protein 24 hr post transfection.