Before you start
- Lyse cells gently and keep everything at 4 °C.
- Plan controls: input (5–10%), isotype IgG IP, and beads-only.
Reagents and materials
Totals are for 4 IPs (+10% extra). Per-IP amounts are in the middle column.
| Reagent | Per IP | Total |
|---|---|---|
| Cell lysate in non-denaturing lysis buffer (0.5–1 mg protein) | 1 mg | 1 mg |
| IP antibody (or matched IgG control) | 2 µg | 2 µg |
| Protein A/G magnetic or agarose beads (slurry) | 25 µL | 25 µL |
| Lysis/wash buffer | 3 mL | 3 mL |
| 2× Laemmli sample buffer | 40 µL | 40 µL |
Buffers and recipes
Non-denaturing IP lysis buffer per 50 mL
| 1 M Tris-HCl, pH 7.4 (50 mM) | 2.5 mL |
| 5 M NaCl (150 mM) | 1.5 mL |
| NP-40 (0.5–1%) | 0.25–0.5 mL |
| 0.5 M EDTA (1 mM) | 0.1 mL |
| Glycerol (5%, optional) | 2.5 mL |
| Ultrapure water | to 50 mL |
Add protease/phosphatase inhibitors fresh. Avoid SDS and deoxycholate, which disrupt interactions.
Equipment
- End-over-end rotator at 4 °C
- Magnetic rack or microcentrifuge
- Western blotting set-up
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Lyse and clear
Lyse cells 30 min on ice in IP buffer, centrifuge 14,000 × g for 10–15 min at 4 °C and keep 5–10% as input.
Pre-clear (optional)
Rotate lysate with beads alone for 30–60 min at 4 °C and discard the beads.
Bind antibody
Add IP antibody (or IgG control) and rotate overnight at 4 °C (or 2–4 h).
Capture
Add protein A/G beads and rotate 1–2 h at 4 °C.
Wash
Wash beads 3–5 times with cold lysis buffer.
Elute
Boil beads in 2× sample buffer for 5 min at 95 °C.
Detect
Run IP, IgG and input samples by western blot for the bait and the partner.
Critical points
- Use light-chain- or conformation-specific secondary antibodies to avoid IgG heavy/light chain bands at 50 and 25 kDa.
- An interaction should be absent from the IgG control and ideally confirmed by reciprocal IP.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Bait not pulled down | Check antibody is IP-validated; increase antibody or lysate; check bead type (A vs G) for the antibody species. |
| High background | More/stringent washes (raise NaCl to 300 mM), pre-clear. |
Safety
Standard laboratory care.
Further reading
- Current Protocols in Protein Science: immunoprecipitation.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.