Immunoprecipitation
Protocol
- This step pre-clears crude cell extract of proteins which can bind non-specifically to the beads. In a 1.5 ml microcentrifuge tube, add 25 μl Protein A Magnetic Beads to 200μl of crude cell extract. Gently vortex andincubate 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. Discard beads.
- Add 1-5 μg of antibody to crude cell lysate vortex and incubate at 4°C for 1 hour. (If monoclonal antibodies are used, add 5 μg rabbit anti-mouse IgG antibody. Vortex and incubate an additional 30 minutes at 4°C).*
*Alternatively, Protein G Magnetic Beads (NEB #S1430S) can be used for immunoprecipitations with monoclonal antibodies. - Add 25 μl of Protein A Magnetic Beads suspension. Gently vortex and 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 with 500 μl of Immunoprecipitation Buffer by gentle vortex. Apply magnetic field then remove supernatant and discard. Repeat wash 2 times.
- Resuspend bead pellet in 30 μl of 3X SDS Sample Loading Buffer (187.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 150 mM DTT, 0.03% (w/v) bromophenol blue, 2% β-mercaptoethanol).
- Incubate sample at 70°C for 5 minutes.
- Apply magnetic field to sample then load supernatant on SDS-PAGE gel and electrophorese.
