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 bufferUse 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 sampleUse 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 lysateAdd 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 targetAdd 2-5 ug EpiQuik Methylated DNA Immunoprecipitation Kit 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 complexesAdd 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 beadsWash 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 targetFor 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 eluateRun 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 conditionsPrepare 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 gentlyUse 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-clearPre-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 antibodyAdd 2-5 ug EpiQuik Methylated DNA Immunoprecipitation Kit 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 complexesAdd 20-40 uL protein A/G beads and rotate 1-2 hours at 4 degrees C. Keep all steps cold. 6 Wash gentlyWash 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 detectElute with SDS sample buffer for Western blot. Detect bait and suspected partner proteins on separate blots or after stripping when appropriate. 8 Confirm interactionInclude 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. 1 Crosslink sampleTreat 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 lysateCollect cells and prepare nuclei or chromatin-enriched lysate using a compatible buffer. Keep samples cold and include protease inhibitors. 3 Fragment chromatin or complexesSonicate 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-clearClarify lysate and pre-clear with blocked protein A/G or ChIP-compatible beads for 30-60 minutes at 4 degrees C. 5 Add antibodyAdd 2-10 ug EpiQuik Methylated DNA Immunoprecipitation Kit per IP depending on target abundance and chromatin amount. Rotate overnight at 4 degrees C for chromatin-associated targets. 6 Capture and washAdd 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 crosslinksElute 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 analyzePurify 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.
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. 1 Prepare RNase-free workspaceUse RNase-free tubes, filter tips, buffers, and gloves. Add RNase inhibitor to lysis, wash, and binding buffers when compatible. 2 Lyse cells gentlyLyse 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 lysatePre-clear lysate with washed beads for 30-60 minutes at 4 degrees C. Transfer supernatant to a new RNase-free tube. 4 Add antibodyAdd 2-10 ug EpiQuik Methylated DNA Immunoprecipitation Kit 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 complexesAdd 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 conditionsWash 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 RNADigest 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 RNAAnalyze 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.
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