Immunoprecipitation Protocol: A Visual Guide | Cell Signaling Technology

Learn the principles of immunoprecipitation for enriching proteins from cell or tissue lysates. Review guidelines for selecting antibodies, magnetic beads, and appropriate controls (Input, isotype, beads-only) to optimize IP experiments.

Prepare 1× PBS on ice, make 1× cell lysis buffer with PMSF at final concentration of 1 mM, and prepare 3× SDS sample buffer with DTT. Keep all solutions on ice before use.

Treat cells with activators or inhibitors if needed, wash with ice-cold PBS, and lyse with cell lysis buffer on ice for 5 minutes. Scrape cells and transfer to microcentrifuge tubes, then sonicate three times for 5 seconds each with a probe sonicator.

Centrifuge sonicated lysate at 14,000×g for 10 minutes at 4°C to pellet cell debris. Transfer supernatant to new tube and reserve a portion as Input control. Pre-wash magnetic or agarose beads with cell lysis buffer according to bead type.

Incubate lysate with unconjugated beads (without antibody) to remove proteins with non-specific bead binding. Centrifuge or magnetically separate beads, then transfer clarified lysate to new tube for immunoprecipitation reaction.

Add appropriate antibody at recommended dilution to lysate and incubate overnight at 4°C on a rotator. Add pre-washed beads (Protein A/G agarose, biotin-streptavidin, or antibody-conjugated beads) and incubate 1-3 hours or overnight at 4°C depending on bead type.

Separate beads by centrifugation or magnetic separation, save supernatant (unbound proteins) if needed, then wash pellet 5 times with 500 µL cell lysis buffer. Resuspend final bead pellet in 20-40 µL 3× SDS sample buffer and heat at 95-100°C for 5 minutes.

Centrifuge or magnetically separate beads to pellet them away from protein-containing supernatant. Load 15-30 µL of supernatant onto SDS-PAGE gel along with protein marker, Input control, and unbound protein samples, then proceed with Western blot detection.