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Chapters
- 00:05Title
- 01:38Purification of GTPase-binding Proteins
- 04:45Nucleotide Loading of GTPase
- 06:32Competition Binding
- 08:42Results: Determination of the Relative Binding Affinities of RCC2 and Coronin-1C for Rac1
- 10:03Conclusion
This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.
