Immunoprecipitation and GST pull-down

Co-immunoprecipitations and pull-down assays

For each IP:

-3-4 μg of the appropriate antibody (typically 15 μl of a santa cruz Ab)

-12 μl of protein A sepharose beads (Amersham) or Protein A agarose (Repligen)

-400 or 500 μg of proteins are typically used. Or 100 μl of 293 cell lysate. See below for lysate preparation.

-Add IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween-20, 1% Igepal 30 (a.k.a. NP-40); complemented with 1 mM dithiothreitol, 10 mM b-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 10 mg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin-A, and 1 mM phenylmethylsulfonylfluoride) to a final volume of 500-600 μl.

-incubate for 3 h at 4°C.

-Immunoprecipitates are rinsed 3 times in cold IP buffer (to rinse, spin tubes at 1000rpm 1 min, aspirate the supernatant with a protein gel loading tip, or with a pipetman, without aspirating the beads, add 600 μl of IP buffer, vortex shortly, and repeat)

-Add 10 μl of 4X sample buffer to the bead pellet and boil 5 min before loading onto SDS-PAGE for western blotting.

For Pull-down assays simply replace the antibody and protein A beads by 10-15 μl of GST-fusion protein beads (using GST only-beads as a negative control) and follow the same protocol. After transfer of gel, coomassie stain gel to have bead input.

Keep an aliquot of lysate (40-80 μg protein/well) to run on a gel and blot for proteins of interest to determine what the input was in the IP.

For lysate preparation:

-Put chunk of tissue or cell pellet in a suitable volume of IP buffer (depends on how much you have, but typically a protein concentration of 2-5 μg/μl of protein is good). 

For 293 cells, lyse a P100 plate with 800 μl lysis buffer, scrape cells and put into eppendorf tube.

-Sonicate briefly (for 10 sec) to disrupt tissue or cells.

-Spin 5 min at 14,000 rpm at 4°C. 

-Keep supernatant and discard pellet.

-Freeze at –80°C

-Do a Bradford protein assay (Biorad) to determine protein concentration. Not needed for 293 cell lysate

Western blotting

Run SDS-PAGE

Transfer onto PVDF or nitrocellulose membranes.

Block membranes in PBS containing 0.1% Tween-20 and 10% milk for 1 h. 

Incubated with primary antibodies overnight at 4°C

Rinse 3x 5 min in PBS-0.1% Tween 20

Incubate with secondary antibodies for 2 h at room temperature. 

All antibodies are diluted in PBS containing 0.1% Tween-20 and 5% milk. 

ECL (Amersham) for immunodetection.

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