Immunoprecipitation using Protein A/G Magnetic Beads
Rinse a 60 mm culture dish of confluent cells with PBS.
Lyse the cells with 0.5 ml cold Immunoprecipitation Buffer.
Maintain constant agitation for 30 minutes at 4°C.
Scrape the cells from the dish.
Sonicate on ice for 5 seconds; repeat 4 times.
Centrifuge for 5 minutes at 4°C.
Assay for total protein then adjust concentration to approximately 1 mg/ml with Immunoprecipitation Buffer.
In a 1.5 ml microcentrifuge tube, add 25 μl Protein A/G Magnetic Beads to 200 μl of crude cell extract.
Gently vortex.
Incubate at 4°C for 1 hour.
Apply magnetic field for 30 seconds to pull beads to the side of the tube.
Pipette supernatant to a clean 1.5 ml microcentrifuge tube and discard the beads.
Add 1-5 μg of antibody to crude cell lysate.
Vortex.
Incubate at 4°C for 1 hour.
Add 25 μl of Protein A/G Magnetic Beads suspension.
Gently vortex.
Incubate with agitation for 1 hour at 4°C.
Apply magnetic field to pull beads to the side of the tube.
Carefully pipette to remove supernatant.
(wash #1) Wash with 500 μl of Immunoprecipitation Buffer by gentle vortex.
(wash #1) Apply magnetic field then remove supernatant and discard.
(wash #2) Wash with 500 μl of Immunoprecipitation Buffer by gentle vortex.
(wash #2) Apply magnetic field then remove supernatant and discard.
(wash #3) Wash with 500 μl of Immunoprecipitation Buffer by gentle vortex.
(wash #3) Apply magnetic field then remove supernatant and discard.
Resuspend bead pellet in 30 μl of 3X SDS Sample Loading Buffer.
Incubate sample at 70°C for 5 minutes.
Apply magnetic field to sample then load supernatant on SDS-PAGE gel and electrophorese.
