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.
