Last reviewed: 18 October 2026.
Immunoprecipitation (IP), co-immunoprecipitation (Co-IP), pull-down assays and chromatin immunoprecipitation (ChIP) all use an affinity reagent to pull a target out of a complex lysate. This guide explains when to use each, gives a step-by-step IP workflow, and lists the controls and critical parameters that decide whether the result can be trusted.
Key points
- IP isolates one protein with an antibody; Co-IP isolates the protein together with its binding partners; a pull-down uses a tagged bait protein (for example GST- or His-tagged) instead of an antibody; ChIP isolates protein–DNA complexes after cross-linking.
- The antibody must be validated for IP: IP recognises native protein, so an antibody that works in Western blot may fail in IP.
- Protein A binds rabbit IgG strongly; Protein G binds a broader range including most mouse IgG subclasses; Protein A/G beads cover both.
- Use mild, non-denaturing lysis buffers for Co-IP to keep complexes intact; stronger detergent (RIPA-type) buffers suit single-protein IP.
- Every IP needs an isotype or beads-only control and an input sample.
- Typical ChIP fixation is 1% formaldehyde for about 10 minutes, quenched with 125 mM glycine.
- Chromatin is sheared to roughly 200–1000 bp; sonication conditions must be optimised for each cell type and instrument.
- Longer sonication gives smaller fragments and higher resolution but lower recovery; shorter sonication does the opposite.
IP, Co-IP, pull-down or ChIP: which one do I need?
| Question | Method | Capture reagent | Read-out |
|---|---|---|---|
| Is my protein present, modified or cleaved? | IP | Antibody to the target | Western blot, mass spectrometry |
| Which proteins bind my protein in cells? | Co-IP | Antibody to the target | Western blot for the partner, mass spectrometry |
| Do two proteins bind directly in vitro? | Pull-down | Tagged recombinant bait on glutathione or Ni-NTA beads | Western blot for the prey |
| Where on the genome does my protein bind? | ChIP | Antibody to the DNA-binding protein or histone mark | qPCR, sequencing (ChIP-seq) |
Immunoprecipitation protocol, step by step
- Lyse cells on ice in a buffer with protease and phosphatase inhibitors; use a non-denaturing buffer (for example NP-40 or Triton X-100 based) for Co-IP. Clear the lysate by centrifugation.
- Keep an input sample (a small fraction of the lysate) for later comparison.
- Pre-clear (optional) by incubating lysate with beads alone to remove proteins that bind beads non-specifically.
- Incubate with antibody at 4 °C, typically 1 hour to overnight with gentle rotation. In parallel, set up an isotype-control antibody of the same host and class.
- Capture the antibody–antigen complex on Protein A, G or A/G beads (agarose or magnetic), typically 1–4 hours at 4 °C.
- Wash the beads several times with lysis or wash buffer; more stringent washes lower background but can strip weak interactions in Co-IP.
- Elute by boiling in SDS sample buffer (for Western blot) or with low-pH glycine or competing peptide (to keep proteins native).
- Analyse input, IP and control lanes side by side by Western blot. To avoid detecting the IP antibody’s heavy (~50 kDa) and light (~25 kDa) chains, use a detection antibody from a different species or a light-chain-specific secondary.
How is a pull-down assay different?
A pull-down replaces the antibody with a purified, tagged bait protein. GST-tagged bait is immobilised on glutathione beads and His-tagged bait on Ni-NTA beads; cell lysate or a purified prey protein is passed over the beads, washed and eluted. The essential control is the tag alone (for example GST protein on the same beads), which shows how much prey binds the tag or beads rather than the bait.
Chromatin immunoprecipitation (ChIP), step by step
- Cross-link cells with 1% formaldehyde, typically about 10 minutes, then quench with 125 mM glycine for about 5 minutes.
- Lyse cells and isolate nuclei.
- Shear chromatin by sonication (or micrococcal nuclease digestion) to fragments of roughly 200–1000 bp. Run pilot shearing and check fragment size on an agarose gel after reversing cross-links.
- Immunoprecipitate with a ChIP-validated antibody, alongside an IgG control and an input sample.
- Wash with low-salt, high-salt and LiCl buffers, then elute.
- Reverse cross-links by heating, digest RNA and protein, and purify DNA.
- Quantify by qPCR (percent of input at target and control regions) or sequencing.
Which controls make an IP or ChIP result credible?
- Input: confirms the target is present in the starting lysate and normalises ChIP-qPCR.
- Isotype IgG or beads-only: shows non-specific binding to antibody or beads.
- Negative genomic region (ChIP): a region where the protein should not bind.
- Knockout or knockdown cells: the strongest specificity control for the IP antibody.
Antibodies for IP and ChIP
Choose antibodies whose datasheets list IP (or ChIP) among validated applications. RayBiotech datasheets state validated applications per catalog number; for example RayBiotech anti-CD80 DS-MB-03212 lists flow cytometry, immunoprecipitation and Western blotting. For detecting interaction partners or secreted factors downstream, see RayBiotech antibody arrays and ELISA kits. For a guide to choosing primary and secondary antibodies, see primary and secondary antibody selection for Western blot.
Sources
- Rockland Immunochemicals. Chromatin immunoprecipitation (ChIP) protocol. rockland.com
- Stanford University, Gozani lab. Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences. PDF
- Published ChIP method using 1% formaldehyde for 10 min and 125 mM glycine quench. PMC3201960
- RayBiotech product datasheets for the catalog numbers cited above. raybiotech.com
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