Immunoprecipitation Protocol
Collect cells and centrifuge at 1200 rpm for 5 minutes at 4°C .
Wash protein G agarose beads with cell lysis buffer by pulsing in a microcentrifuge tube ( two minutes at 5,000 rpm ) .
Aspirate and discard supernatant .
Wash the beads with celllysis buffer ( 1 / 3 ) .
Wash the beads with celllysis buffer ( 2 / 3 ) .
Wash the beads with celllysis buffer ( 3 / 3 ) .
Adjust antibody concentration to 5 - 10 µg / ml in PBS and transfer 500 µl of diluted antibody to 5 - 10 µl of agarose beads for each sample .
Place the antibody - protein G agarose mix on a shaker and rotate at 4°C for one hour .
Spin down the protein G beads for two minutes at 5,000 rpm and wash the antibody - beads three times with cell lysis buffer .
Discard the supernatant and immediately add 800 µl of ice - cold lysis buffer to the cells and vortex , then incubate for 30 minutes on ice .
Freeze and thaw the samples with dry ice for two more cycles or sonicate for 15 seconds to ensure the full release of the proteins from the cells .
Spin lysates at 14,000 rpm in a pre - cooled centrifuge for 10 minutes and keep the supernatant .
Adjust the protein concentration of the supernatant to 1 - 2 mg / ml with lysis buffer .
Mix 100 - 500 µl of cell extract with antibody - protein G agarose and rotate the samples at 4°C for about two hours .
Collect the agarose beads by pulsing in a microcentrifuge tube ( two minutes at 5,000 rpm , 4°C ) .
Aspirate and discard the supernatant .
Wash the beads with ice - cold cell lysis buffer ( 1 / 3 ) .
Wash the beads with ice - cold cell lysis buffer ( 2 / 3 ) .
Wash the beads with ice - cold cell lysis buffer ( 3 / 3 ) .
After the final wash , remove the supernatant and add 20 µl of 2X SDS sample buffer .
Load 30 µl of sample in each well of a 1.5 mm thick gel .
Run the gel according tomanufacturer’s recommendations and continue with immunoblotting using BioLegend’s Western Blotting protocol ( alternately , radiolabeled proteins prepared from target cells can beused to directly visualize the immunoprecipitated protein ) .
Boil for 5 minutes at 95°C .
Carefully pipette off the supernatant .
Spin down the beads at maximum speed in a microcentrifuge for 5 minutes at room temperature .
