Determining Rho GTPase Activity by an Affinity-Precipitation Assay

Abstract

The Rho GTPases are members of the Ras superfamily of GTPases that are pivotal regulators of the actin cytoskeleton. They also contribute to other cellular processes such as gene transcription, cell polarity, microtubule dynamics, cell cycle progression and vesicle trafficking. Most Rho GTPases act as molecular switches cycling between an "active" GTP-bound form and an "inactive" GDP-bound form. Hence, to elucidate the mechanisms by which Rho GTPases regulate cellular responses, an important parameter to determine is the GTP-loading of each Rho family member in cells under different conditions. Here we describe a biochemical technique to assess this based on affinity-precipitation of the GTP-bound form from whole cell lysates.

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“…Small GTPase activity was assessed by GTPase affinity precipitation as previously described [ 11 ]. Briefly, cells were lysed in MLB buffer (Millipore, Bedford, MA, USA), and the resulting crude protein were reacted with Rac/Cdc42-binding domain and Rho protein-binding domain agarose conjugate beads.…”