Chromatin Immunoprecipitation Assay (ChIP) Detailed Protocol (For Reference Only)

Chromatin Immunoprecipitation (ChIP) is a core technique for studying protein-DNA interactions. It involves cross-linking DNA-protein complexes in cells with formaldehyde, enriching chromatin fragments bound to target proteins using specific antibodies, and after de-cross-linking and DNA purification, detecting the DNA sequences bound by the target protein via qPCR or sequencing (ChIP-seq). This method enables precise analysis of epigenetic regulatory mechanisms such as transcription factor binding sites and histone modification distribution. The detailed experimental steps are as follows:

I. Pre-Experimental Preparation

1. Reagent Preparation

Basic Reagents: PBS buffer (pH 7.4, pre-cooled), 16% formaldehyde solution (for cross-linking, freshly prepared), glycine solution (1.25M, for terminating cross-linking), cell lysis buffer (supplemented with protease inhibitors), nuclear lysis buffer (supplemented with protease inhibitors and SDS), ChIP wash buffers (low-salt buffer, high-salt buffer, lithium chloride buffer, TE buffer, all pre-cooled), elution buffer (containing 1% SDS and 0.1M NaHCO₃), NaCl solution (5M, for de-cross-linking), proteinase K solution (20mg/mL), RNase A solution (10mg/mL), phenol-chloroform-isoamyl alcohol mixture (25:24:1), absolute ethanol, 75% ethanol, TE buffer (pH 8.0, containing RNase A).

Core Reagents: Specific primary antibody against the target protein (e.g., transcription factor antibody, histone modification antibody, validated for ChIP compatibility), negative control antibody (e.g., irrelevant IgG), positive control antibody (e.g., RNA polymerase Ⅱ antibody), Protein A/G magnetic beads (pre-washed and blocked, for capturing antibody-chromatin complexes).

2. Equipment Preparation

Refrigerated centrifuge (capable of 12,000×g), sonicator (equipped with cell disruption probe), pipettes and tips (RNase/DNase-free), centrifuge tubes (1.5mL/15mL, RNase/DNase-free), magnetic rack, orbital shaker (4℃), water bath, fume hood, electrophoresis system, gel documentation system, qPCR instrument (or high-throughput sequencing platform).

3. Sample Pretreatment

Cell Samples: Culture cells to the logarithmic growth phase, adjust the cell density to 1×10⁷ cells/sample. Collect cells in 15mL centrifuge tubes, centrifuge at 300×g for 5 minutes at 4℃, discard the supernatant. Wash twice with pre-cooled PBS, then resuspend in 1mL PBS (maintain cell viability).

Tissue Samples: Take fresh tissue (50-100mg), rinse with pre-cooled PBS to remove blood impurities, mince into small pieces, add 5mL PBS, and homogenize to form a single-cell suspension. Centrifuge at 300×g for 5 minutes at 4℃, discard the supernatant, wash twice with PBS, and resuspend in 1mL PBS.

II. Cross-Linking Fixation (Preserve DNA-Protein Interactions)

Add 16% formaldehyde solution to the cell suspension to a final concentration of 1%, gently invert to mix, and incubate at room temperature for 10 minutes (cross-linking time needs optimization to avoid excessive cross-linking, which may hinder sonication).

Add 1.25M glycine solution to a final concentration of 0.125M, incubate at room temperature for 5 minutes to terminate the cross-linking reaction.

Centrifuge at 300×g for 5 minutes at 4℃, discard the supernatant. Wash the cells twice with pre-cooled PBS, centrifuge to collect cells after each wash, and finally discard all PBS to obtain cross-linked cell pellets.

III. Cell Lysis and Nuclear Isolation

Add 1mL cell lysis buffer (supplemented with protease inhibitors) to the cell pellet, incubate on ice for 10 minutes to lyse the cytoplasmic membrane.

Centrifuge at 600×g for 5 minutes at 4℃, discard the supernatant, and collect the nuclear pellet (semi-transparent flocculent precipitate).

Add 300μL nuclear lysis buffer (containing 1% SDS and protease inhibitors) to the nuclear pellet, gently pipette to mix, and incubate on ice for 10 minutes to lyse the nuclear membrane and release chromatin (the solution will become viscous).

IV. Chromatin Sonication (Obtain Target-Length Fragments)

Transfer the nuclear lysate to a dedicated sonication tube and place it in an ice bath (maintain the sample at low temperature during sonication to avoid protein degradation).

Set sonication parameters (need pre-experiment optimization, e.g., power 30%, sonication 10s, interval 20s, 10-15 cycles) to fragment chromatin into 200-500bp segments (this length is suitable for subsequent immunoprecipitation and PCR amplification).

After sonication, centrifuge at 12,000×g for 15 minutes at 4℃, collect the supernatant (containing sheared chromatin fragments), which is the ChIP sample stock solution.

Take 5μL of the stock solution, add 1μL RNase A, incubate at 37℃ for 30 minutes, then add 5μL 6×DNA loading buffer. Verify the fragment length by 1% agarose gel electrophoresis (target bands should be concentrated at 200-500bp).

V. Immunoprecipitation (Enrich Chromatin Bound to Target Protein)

Take 200μL of the chromatin stock solution, add 800μL ChIP dilution buffer (reduce SDS concentration to avoid affecting antibody binding), gently mix to obtain the chromatin dilution.

Take 10μL of the chromatin dilution as the "Input control" (for calculating immunoprecipitation efficiency), add 90μL TE buffer, and store at -20℃ for later use.

Add 5μg specific primary antibody against the target protein to the remaining chromatin dilution. For the negative control tube, add 5μg irrelevant IgG; for the positive control tube, add 5μg positive control antibody. Incubate overnight (12-16 hours) on a slow orbital shaker at 4℃ to ensure sufficient binding between the antibody and target protein.

Pre-wash Protein A/G magnetic beads three times with ChIP wash buffer in advance, resuspend in 50μL wash buffer, add the bead suspension to each sample tube, and incubate on an orbital shaker at 4℃ for 2 hours to capture antibody-chromatin complexes.

Place the sample tubes on a magnetic rack to adsorb the beads, discard the supernatant, and retain the bead-complex precipitate.

VI. Multi-Round Washing (Remove Non-Specific Binding Impurities)

Add 1mL pre-cooled low-salt buffer to the bead precipitate, gently invert to mix, incubate at room temperature for 5 minutes, place on the magnetic rack to adsorb the beads, and discard the supernatant.

Add 1mL pre-cooled high-salt buffer, repeat the washing step (incubate at room temperature for 5 minutes, adsorb on the magnetic rack, and discard the supernatant).

Add 1mL pre-cooled lithium chloride buffer, repeat the washing step.

Add 1mL pre-cooled TE buffer, repeat the washing step (4 rounds of washing in total to ensure removal of unbound chromatin, free antibodies, and other impurities).

After the final wash, aspirate as much residual supernatant as possible (avoid impurity interference in subsequent experiments).

VII. Elution and De-Cross-Linking (Release Target DNA Fragments)

Add 250μL elution buffer to the bead precipitate, gently pipette to mix, incubate at room temperature for 15 minutes (gently invert to mix every 5 minutes during incubation to promote complex elution).

Place on the magnetic rack to adsorb the beads, transfer the eluate to a new centrifuge tube (the eluate contains antibody-protein-DNA complexes).

Add 25μL 5M NaCl to the Input control sample and 10μL 5M NaCl to the eluate samples. Incubate in a 65℃ water bath for 4 hours (or overnight) to fully de-cross-link DNA and proteins.

Add 1μL RNase A (10mg/mL) to each sample, incubate at 37℃ for 30 minutes to degrade RNA impurities.

Add 1μL proteinase K solution (20mg/mL), incubate at 45℃ for 1 hour to degrade proteins (including antibodies, target proteins, and enzymes).

VIII. DNA Purification and Recovery (Obtain Pure Target DNA)

Add an equal volume of phenol-chloroform-isoamyl alcohol mixture (25:24:1) to each sample, vortex vigorously for 1 minute, and centrifuge at 12,000×g for 10 minutes at 4℃.

Carefully aspirate the upper aqueous phase (containing DNA) into a new centrifuge tube, avoiding the intermediate protein layer.

Add an equal volume of chloroform, vortex for 30 seconds, centrifuge at 12,000×g for 10 minutes at 4℃, and aspirate the upper aqueous phase again (to further remove phenol impurities).

Add 2 volumes of absolute ethanol and 1/10 volume of 3M NaAc (pH 5.2) to the aqueous phase, gently invert to mix, and incubate at -20℃ for 30 minutes (or overnight) to precipitate DNA.

Centrifuge at 12,000×g for 15 minutes at 4℃, discard the supernatant to obtain the DNA pellet.

Add 1mL 75% ethanol, gently invert to wash the DNA pellet, centrifuge at 12,000×g for 5 minutes at 4℃, and discard the supernatant.

Invert the centrifuge tube at room temperature to air-dry the DNA pellet (avoid over-drying, which may make DNA difficult to dissolve). Add 20-50μL TE buffer (containing RNase A) and dissolve overnight at 4℃ (or incubate at 37℃ for 30 minutes to accelerate dissolution).

Detect DNA concentration and purity using a Nanodrop (A260/A280 ratio should be between 1.8-2.0 to ensure DNA purity).

IX. Detection and Analysis (Verify Target DNA Fragments)

1. qPCR Detection (Quantitative Analysis of Binding Efficiency)

Design specific primers for the target gene binding region (e.g., sequences upstream/downstream of the transcription factor binding site), and design primers for irrelevant regions as negative controls.

Perform qPCR reactions using purified IP sample DNA and Input control DNA as templates (reaction system: 10μL 2×qPCR Mix, 0.5μL each primer, 2μL DNA template, 7μL ddH₂O).

Reaction program: 95℃ pre-denaturation for 5 minutes; 40 cycles of 95℃ denaturation for 10s, 60℃ annealing for 30s, 72℃ extension for 30s; melting curve analysis to verify primer specificity.

Calculate immunoprecipitation efficiency: Use the 2^(-ΔΔCt) method, with the Input control as the reference, to compare the enrichment fold of the target region between the IP sample and the negative control (IgG group) (enrichment fold > 2x is considered effective binding).

2. Sequencing Analysis (ChIP-seq, Genome-Wide Screening of Binding Sites)

Construct libraries from purified DNA samples (end repair, A-tailing, adapter ligation, PCR amplification).

After passing library quality control, perform high-throughput sequencing (e.g., Illumina platform) to obtain raw sequencing data.

Data processing: Filter raw data, align to the reference genome, peak calling (e.g., MACS2 software), and screen target protein binding peaks.

Bioinformatics analysis: Annotation of binding peaks (promoters, enhancers, etc.), GO/KEGG functional enrichment analysis, Motif analysis (predict target protein binding motifs).

Key Notes：

Cross-linking is the core of the experiment: Strictly control the final concentration of formaldehyde at 1%, and avoid excessively long incubation time (5-10 minutes) to prevent incomplete chromatin sonication due to over-cross-linking; ensure sufficient glycine termination to avoid residual formaldehyde affecting subsequent reactions.

Sonication conditions require individual optimization: Chromatin hardness varies among different cells/tissues. Adjust sonication power, time, and cycles through pre-experiments to ensure fragment lengths are concentrated at 200-500bp (excessively long or short fragments will affect IP efficiency).

Antibody selection is crucial: Use specific antibodies validated for ChIP (avoid antibodies only suitable for WB/IHC). The negative control (IgG) should match the species of the primary antibody, and the positive control is used to verify the effectiveness of the experimental system.

Inhibit nucleases and proteases throughout the process: Freshly add protease inhibitors to all lysis buffers and wash buffers. Maintain samples at low temperature (on ice or 4℃) during processing to avoid DNA degradation and protein denaturation.

Washing steps should be thorough but gentle: Multi-round washing can reduce background, but operations should be gentle to avoid detachment of bead-complexes due to vigorous vortexing; aspirate as much supernatant as possible after the final wash to reduce impurity residues.

Ensure complete de-cross-linking: Incubate at 65℃ for no less than 4 hours to fully separate DNA and proteins, otherwise DNA purification efficiency and subsequent detection results will be affected.

Exercise caution during DNA purification: Avoid aspirating the intermediate protein layer during phenol-chloroform extraction; add glycogen (10μg/sample) during ethanol precipitation to improve the recovery rate of low-concentration DNA.

Experimental controls are indispensable: Input control is used to calculate enrichment efficiency, negative control (IgG) to exclude non-specific binding, and positive control to verify the stability of the experimental system—all three are essential.