Cart (0)
Choose Your IP Workflow
Use this immunoprecipitation protocol guide to enrich a target protein, preserve protein complexes, handle crosslinked samples, or perform RNA immunoprecipitation using antibody-based capture.
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 your own non-denaturing IP-compatible lysis buffer or a buffer selected from the Western blot protocol that matches the sample type and target. As a starting point, use 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 such as Protease Inhibitor Cocktail when compatible with the downstream assay. 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 with cold lysis buffer containing fresh protease inhibitors. Incubate 15-30 minutes on ice 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.
Co-IP protocol
Use this workflow to preserve and test protein-protein interactions during immunoprecipitation.
- Use this format when: Pulling down a bait protein and detecting associated partner proteins.
- Optimize first: Mild lysis conditions, salt concentration, detergent choice, lysate freshness, wash stringency, and reciprocal IP controls.
1
Use mild lysis conditions
Prepare cold mild lysis buffer such as 20-50 mM Tris or HEPES, 100-150 mM NaCl, 0.2-0.5 percent NP-40 or digitonin, 1 mM EDTA, and fresh inhibitors. Avoid harsh detergents and high salt unless the interaction is strong.
2
Lyse gently
Use fresh cells when possible. Lyse on ice for 15-30 minutes with gentle mixing. Clarify lysate at 10,000-14,000 x g for 10 minutes at 4 degrees C. Avoid sonication unless necessary because it can disrupt complexes.
3
Pre-clear
Pre-clear lysate with 20-30 uL beads for 30-60 minutes at 4 degrees C. Transfer the supernatant to a new tube.
4
Add bait antibody
Add 2-5 ug primary antibody per IP if it is the bait antibody. Rotate 2-4 hours at 4 degrees C. For weak interactions, overnight incubation can help but may increase nonspecific binding.
5
Capture complexes
Add 20-40 uL protein A/G beads and rotate 1-2 hours at 4 degrees C. Keep all steps cold.
6
Wash gently
Wash 3-4 times with mild lysis buffer. If nonspecific bands are high, increase salt stepwise to 200-300 mM NaCl or reduce detergent exposure based on complex stability.
7
Elute and detect
Elute with SDS sample buffer for Western blot. Detect bait and suspected partner proteins on separate blots or after stripping when appropriate.
8
Confirm interaction
Include IgG control, beads-only control, input lysate, and reciprocal Co-IP when possible. Validate that the partner signal is enriched in the bait IP compared with controls.
Complex preservationCo-IP success depends heavily on keeping the complex intact. Use fresh lysate, cold buffers, and mild washing first.
Nuclease optionFor DNA- or RNA-mediated associations, add nuclease controls when needed to distinguish direct protein interaction from nucleic-acid bridged association.
Crosslinked IP or chromatin-associated target protocol
Use this workflow as a general crosslinked IP starting point for chromatin-associated proteins. For full ChIP workflows, see the dedicated ChIP protocol.
- Use this format when: A chromatin-associated protein or complex needs crosslink-stabilized enrichment before downstream analysis.
- Optimize first: Crosslinking time, chromatin fragmentation, antibody amount, wash stringency, reversal conditions, and DNA/protein recovery method.
- Alternative workflow: If the goal is target-specific protein-DNA enrichment rather than conventional crosslinked IP or ChIP, consider EpiNext CUT&LUNCH Assay Kit or EpiNext CUT&RUN-Fast Kit.
1
Crosslink sample
Treat cells with 1 percent formaldehyde for 10 minutes at room temperature as a starting condition. Quench with 125 mM glycine for 5 minutes. Wash cells with cold PBS containing inhibitors.
2
Prepare nuclei or lysate
Collect cells and prepare nuclei or chromatin-enriched lysate using a compatible buffer. Keep samples cold and include protease inhibitors.
3
Fragment chromatin or complexes
Sonicate or digest to produce the fragment size needed for the downstream assay. For ChIP-qPCR, 200-800 bp DNA fragments are often used. Confirm shearing before large experiments.
4
Clarify and pre-clear
Clarify lysate and pre-clear with blocked protein A/G or ChIP-compatible beads for 30-60 minutes at 4 degrees C.
5
Add antibody
Add 2-10 ug primary antibody per IP depending on target abundance and chromatin amount. Rotate overnight at 4 degrees C for chromatin-associated targets.
6
Capture and wash
Add blocked beads and rotate 1-2 hours. Wash sequentially with low-salt, high-salt, LiCl or detergent wash, and TE buffer when compatible with the downstream method.
7
Elute and reverse crosslinks
Elute complexes and reverse crosslinks, commonly at 65 degrees C for 2-6 hours or overnight depending on protocol. Treat with RNase and proteinase K if recovering DNA.
8
Purify and analyze
Purify DNA or protein as required. Analyze by qPCR, sequencing library preparation, Western blot, or other downstream method.
Dedicated ChIP pageFor optimized native and crosslinking ChIP details, see the dedicated ChIP protocol.
CrosslinkingOver-crosslinking can reduce antibody access and recovery. Start with mild crosslinking and optimize.
CUT&LUNCH or CUT&RUN-Fast optionFor protein-DNA interaction profiling from cells or tissues, use EpiNext CUT&LUNCH Assay Kit or EpiNext CUT&RUN-Fast Kit only when that assay format fits the experimental goal.
RNA immunoprecipitation protocol
Use this workflow to enrich RNA-associated proteins or protein-bound RNA while preserving RNA integrity.
- Use this format when: Studying RNA-binding proteins, protein-RNA complexes, or RNA associated with a target protein.
- Optimize first: RNase-free handling, lysis conditions, antibody amount, salt concentration, wash stringency, and RNA purification.
- Kit option: For a complete RIP workflow with coordinated reagents, use EpiNext CUT&LUNCH RNA Immunoprecipitation (RIP) Kit when its sample type and downstream readout match the experiment.
1
Prepare RNase-free workspace
Use RNase-free tubes, filter tips, buffers, and gloves. Add RNase inhibitor to lysis, wash, and binding buffers when compatible.
2
Lyse cells gently
Lyse cells in RIP lysis buffer containing mild detergent, physiological salt, protease inhibitors, and RNase inhibitor. Use fresh lysate when possible. Clarify at 12,000-16,000 x g for 10 minutes at 4 degrees C.
3
Pre-clear lysate
Pre-clear lysate with washed beads for 30-60 minutes at 4 degrees C. Transfer supernatant to a new RNase-free tube.
4
Add antibody
Add 2-10 ug primary antibody per RIP depending on target abundance. Rotate 2-4 hours or overnight at 4 degrees C. Include IgG control and input RNA controls.
5
Capture complexes
Add 20-40 uL protein A/G beads that have been washed and blocked with BSA, yeast tRNA, or other compatible blocking reagent. Rotate 1-2 hours at 4 degrees C.
6
Wash under RNase-free conditions
Wash 4-6 times with cold RIP wash buffer. Increase salt only if nonspecific RNA background is high and the interaction can tolerate it.
7
Recover RNA
Digest protein or reverse crosslinks if used, then purify RNA using phenol/chloroform, column purification, or magnetic bead cleanup. Include DNase treatment when downstream qPCR could detect DNA contamination.
8
Analyze RNA
Analyze enriched RNA by RT-qPCR, sequencing, or other downstream assay. Normalize to input and compare to IgG control.
RNase controlRNA loss is often caused by RNase contamination. Use RNase inhibitor and work quickly on ice.
Input controlSave 1-10 percent input before IP for normalization.
RIP kit optionFor a kit-based RNA immunoprecipitation workflow, consider EpiNext CUT&LUNCH RNA Immunoprecipitation (RIP) Kit when a complete RIP reagent set is preferred.
Immunoprecipitation reference notes
Antibody selectionFor immunoprecipitation, the antibody must recognize the target under the capture condition used for the assay. An antibody that works in Western blot may not necessarily work for native IP, co-IP, ChIP, or RIP.
Bead choiceSelect Protein A, Protein G, mixed Protein A/G, magnetic beads, agarose beads, or conjugated beads based on antibody species, antibody isotype, sample type, and downstream detection needs.
Essential controlsInclude input, unbound or flow-through fraction when useful, IgG control, beads-only control, and a positive or known target sample when possible.
Western blot readoutFor IP-Western blot, antibody heavy chain near 50 kDa and light chain near 25 kDa can interfere with target detection. Use light-chain-specific, conformation-specific, directly conjugated, or clean-blot detection strategies when needed.
Protein-DNA profilingWhen the goal is chromatin-bound protein-DNA enrichment rather than protein pulldown, a dedicated CUT&LUNCH or CUT&RUN-Fast workflow may be more suitable.
RNA IP workflowFor RNA-binding protein or protein-bound RNA enrichment, a dedicated CUT&LUNCH RIP kit can be used when a kit-based RNA immunoprecipitation workflow is preferred.
Common problemsWeak recovery often comes from poor antibody capture, harsh lysis, poor epitope accessibility, target degradation, or insufficient target. High background often comes from sticky lysate, too much antibody, insufficient washing, or bead-binding proteins.
Use these conditions as practical starting points. Follow the product datasheet, kit manual, safety data sheet, and institutional safety requirements when they provide application-specific instructions.
