Protocols
Immunoprecipitation Protocol
Add 2 ml ice cold M-PER reagent containing protease inhibitor to 3x60mm plates of transfected cells and incubate at 4° C for 30 minutes.
Disrupt cells by repeated passage through a 23-gauge needle. Transfer to a microcentrifuge tube.
Pellet cellular debris by centrifugation at 10,000xg for 10 minutes at 4° C. Transfer supernatant to a fresh microcentrifuge tube on ice.
Preclear lysate by adding 2.0 mg of the appropriate control IgG (corresponding to the host species of the primary antibody), together with 40 ml of prewashed Protein G-agarose suspension. Incubate at 4° C for 30 minutes.
Centrifuge at 3,000 rpm for 2 minute at 4° C.
Transfer the supernatant (~750 mg total cellular protein) to a microcentrifuge tube. Add 2mg primary antibody and incubate for 1-2 hours at 4° C. Alternatively, if antibody agarose conjugate is employed, add 30 ml and incubate at 4° C overnight with mixing; skip the next step.
Add 30 ml of prewashed Protein G-agarose suspension. Cap tubes and incubate at 4° C on a rotating device overnight.
Collect immunoprecipitates by centrifugation at 3,000 rpm for 2 minute at 4° C. Carefully aspirate and discard supernatant.
Gently wash pellet 3 times with 1 ml PBS containing 0.5 % NP-40 buffer, each time repeating centrifugation step above.
After final wash, carefully aspirate and discard supernatant and resuspend pellet in 40 ml of 2x SDS electrophoresis sample buffer.
Boil samples for 2-3 minutes and centrifuge samples to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant. Unused samples may be stored at -20° C.
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