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Updated: Mar 12, 2026

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
In Vitro Analysis of Tem1 GTPase Activity and Regulation by the Bfa1/Bub2 GAP
Marco Geymonat1, Adonis Spanos2, Katrin Rittinger2
1Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK. mg604@cam.ac.uk.
Abstract:
Tem1 is a small GTPase that controls the mitotic progression of Saccharomyces cerevisiae through the Mitotic Exit Network. Tem1 activity is tightly controlled in mitosis by Bub2 and Bfa1 and is also regulated by the spindle orientation checkpoint that monitors the correct alignment of the mitotic spindle with the mother-daughter axis. In this chapter we describe the purification of Tem1, Bfa1, and Bub2 and a detailed radioactive filter-binding assay to study the nucleotide binding properties of Tem1 and the role of its regulators Bfa1 and Bub2.
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