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.
Total time
~4–6 hours per experiment (protein purification + nucleotide loading + competition assay)
Steps
1
Purify GTPase-binding proteins from expression system
Express and isolate recombinant Rho-family GTPase-binding proteins (e.g., RCC2, Coronin-1C) using standard protein purification techniques. Ensure high purity for subsequent binding assays.
▶ 01:38
2
Load GTPase with controlled nucleotide state
Exchange nucleotides on purified Rac1 or related GTPase to achieve a defined, stable nucleotide-bound conformation. Control for nucleotide hydrolysis to maintain consistent experimental conditions.
▶ 04:45
3
Perform competition binding assay
Incubate nucleotide-loaded GTPase with multiple binding partners simultaneously to measure competition for the shared binding interface. Quantify relative binding by detecting which protein occupies the GTPase binding site.
▶ 06:32
4
Determine relative binding affinities from competition data
Analyze competition assay results to calculate and compare the relative affinities of RCC2, Coronin-1C, and other binding partners for Rac1. Express results quantitatively to rank binding strength.
▶ 08:42