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 .
