Active GTPase Pulldown Protocol.

Ras and its related small GTPases are important signalling nodes that regulate a wide variety of cellular functions. The active form of these proteins exists in a transient GTP bound state that mediates downstream signalling events. The dysregulation of these GTPases has been associated with the progression of multiple diseases, most prominently cancer and developmental syndromes known as Rasopathies. Determining the activation state of Ras and its relatives has hence been of paramount importance for the investigation of the biochemical functions of small GTPases in the cellular signal transduction network. This chapter describes the most broadly employed approach for the rapid, label-free qualitative and semi-quantitative determination of the Ras GTPase activation state, which can readily be adapted to the analysis of other related GTPases. The method relies on the affinity-based isolation of the active GTP-bound fraction of Ras in cellular extracts, followed by its visualization via western blotting. Specifically, we describe the production of the recombinant affinity probes or baits that bind to the respective active GTPases and the pulldown method for isolating the active GTPase fraction from adherent or non-adherent cells. This method allows for the reproducible measurement of active Ras or Ras family GTPases in a wide variety of cellular contexts.