Fig. 3. pH dependency of G protein association with CSP. Immunoblot analysis showing binding of G_ß and G to CSP-GST and Jdomain-GST immobilized on agarose. Fusion proteins were incubated with hippocampal homogenate (in the absence of ATP) at the indicated pH for 1 hour. The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The nitrocellulose membrane was probed with anti-G_ß monoclonal and anti-G polyclonal (Calbiochem). These results are representative of five experiments. G_ß binding to CSP is very stable over a broad pH range. In contrast, G_ß does not bind to the J domain under any of the conditions examined.