Immunoprecipitation of docking proteins Gab1 or IRS1

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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