Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

To perform the competition binding, set up six microfuge tubes each containing 200 microliters of competition binding buffer. Each tube should also contain 10 microliters of the nucleotide-loaded Rac1 beads and five microliters of the Rac1-binding protein A as a constant binding protein.

To each tube, add 0, 1, 2.5, 5, 10, or 20 microliters of Rac1-binding protein B as the variable binding protein. These volumes assume approximately equal stock concentrations of the constant and variable binding proteins and may need to be adjusted.

Make up the total volume of the binding mixture to 235 microliters by addition of the competition binding buffer. Next, set up a microfuge tube containing 200 microliters of the competition buffer, 10 microliters of experimental nucleotide-loaded Rac1 beads, and 10 microliters of Rac1-binding protein A.

Then, set up the GDP, GTPγS, and no nucleotide control tubes as described in the text protocol. Incubate the tubes for two hours, mixing by inversion at 4 degrees Celsius. Following incubation, wash the beads three times with the competition binding buffer.

Finally, elute the bound proteins in 20 microliters of reducing sample buffer.