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Protein immunoprecipitation protocol
Use this workflow to enrich a target protein from cell or tissue lysate using an antibody and protein A/G magnetic or agarose beads.
- Use this format when: Pulling down a target protein for Western blot, mass spectrometry, enzymatic assay, or interaction analysis.
- Optimize first: Lysis buffer stringency, lysate amount, antibody amount, bead amount, binding time, wash stringency, and elution method.
1
Prepare cold lysis buffer
Use non-denaturing IP lysis buffer such as 20-50 mM Tris or HEPES pH 7.4-7.6, 150 mM NaCl, 1 percent NP-40 or Triton X-100, 1 mM EDTA, and fresh protease inhibitors. Add phosphatase inhibitors for phospho or PTM targets.
2
Lyse sample
Use 0.5-2 mg total protein per IP as a starting range. Lyse cells on ice for 15-30 minutes with periodic mixing. For tissue, homogenize gently in cold lysis buffer. Clarify lysate at 12,000-16,000 x g for 10-15 minutes at 4 degrees C.
3
Pre-clear lysate
Add 20-30 uL washed protein A/G beads per IP to lysate and rotate 30-60 minutes at 4 degrees C. Collect supernatant and discard pre-clear beads to reduce nonspecific binding.
4
Bind antibody to target
Add 2-5 ug primary antibody per 0.5-1 mg lysate as a starting point, or follow the datasheet. Rotate 2-4 hours at 4 degrees C, or overnight for low-abundance targets.
5
Capture immune complexes
Add 20-40 uL washed protein A/G magnetic or agarose beads per IP. Rotate 1-2 hours at 4 degrees C. Use protein A, protein G, or A/G according to antibody species and isotype.
6
Wash beads
Wash beads 3-5 times with 0.5-1 mL cold lysis buffer. For high background, add one higher-salt wash such as 300-500 mM NaCl if the interaction or epitope can tolerate it. Keep beads cold and minimize bead loss.
7
Elute target
For Western blot, add 20-40 uL 1x or 2x SDS sample buffer and heat 70-95 degrees C for 5-10 minutes depending on target stability and bead type. For native elution, use low pH glycine or competing peptide if compatible.
8
Analyze eluate
Run input, flow-through if needed, wash control, IgG control, beads-only control, and IP eluate. For WB detection, use a secondary antibody strategy that minimizes IgG heavy-chain and light-chain interference.
Antibody compatibilityWB validation does not guarantee IP performance. IP requires antibody binding under native or partially native conditions.
Heavy chain interferenceUse crosslinked antibody beads, light-chain-specific secondary antibody, native elution, or antibodies from different species when heavy chain overlaps the target size.
