The phosphorylation levels of Gab1 and IRS1 and their association with signaling molecules can be evaluated after specific immunoprecipitation followed by immunoblotting with appropriate antibodies (Fig. 20.2).
Clarified lysates (0.5-1 mg of proteins) prepared as described earlier are incubated for 3 h at 4° with appropriate antibodies preadsorbed on protein G-Sepharose (4 mg of antibodies/sample). After washes with lysis buffer, immune pellets are resuspended in Laemmli buffer and proteins are separated by SDS-PAGE using a 7.5 or 10% resolving gel and transferred to a polyvinylidene difluoride membrane.
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