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Short answer: Chromatin immunoprecipitation (ChIP) is an assay that uses an antibody to pull a specific protein, such as a transcription factor or a modified histone, out of crosslinked, fragmented chromatin, so the DNA bound to that protein can be identified. The enriched DNA is then read out by qPCR (ChIP-qPCR), sequencing, an array, or an exonuclease-refined variant (ChIP-exo). This page is for researchers and lab staff who need to understand the assay family, how a ChIP-qPCR result is calculated, and what each control does and does not prove. It is a concept reference, not a bench protocol.
Definition and scope
ChIP asks one question: at which DNA sequences was a given protein, or a given histone modification, located in the cells at the time they were fixed? The antibody is the assay. Chromatin is crosslinked so protein-DNA contacts are preserved, fragmented, and immunoprecipitated with the antibody; the DNA recovered is the “ChIP DNA”. A sample of the chromatin set aside before the immunoprecipitation, the input, represents the starting material and is used to normalise the result.
The same enrichment step feeds several readouts, and the names in search results (“ChIP assay”, “ChIP-qPCR”, “ChIP-seq protocol”, “ChIP-exo”) mostly describe the readout, not a different pull-down. This page covers the shared concept and the targeted qPCR readout. For the genome-wide sequencing readout, antibody validation and sequencing quality metrics, see the CASRAI guide to ChIP-seq.
How a ChIP experiment is organised in practice
Reference methods describe the same logical stages: fix cells so protein-DNA contacts are retained, fragment the chromatin (CASRAI covers one common fragmentation instrument in what is a sonicator), immunoprecipitate with the target antibody, reverse the crosslinks, purify the DNA, and quantify it. Each stage has method-specific conditions that belong in a validated protocol from the antibody supplier or the published methods literature, not in a summary.
ChIP-qPCR: how percent input and fold enrichment are calculated
ChIP-qPCR measures enrichment at chosen loci with primers designed against them. Thermo Fisher’s ChIP analysis page and a 2021 MethodsX paper on ChIP-qPCR normalisation describe two analysis approaches, and they are not interchangeable.
| Approach | Calculation as published | Stated assumption or weakness |
|---|---|---|
| Percent input | Adjust the input Ct for its dilution by subtracting log2 of the dilution factor (6.644 for a 1% input, dilution factor 100), then percent input = 100 × 2^(adjusted input Ct − IP Ct). Thermo Fisher notes that typically 1% of the starting chromatin is used as input. | Corrects for both background and the amount of input chromatin. The 2021 MethodsX paper reports it has emerged as a normalisation standard for its reproducibility and accuracy, but notes the traditional version assumes a constant volume of isolate per qPCR; with a constant DNA mass it cannot accurately normalise, and the authors propose a constant-amount alternative that requires DNA quantification and may be limited by low IP yield. |
| Fold enrichment | Fold enrichment = 2^(−ΔΔCt), where ΔCt is the IP Ct minus the mock (IgG or no-antibody) Ct. | Assumes background is reproducible across primer sets, samples and experiments. Thermo Fisher notes this often is not the case, and the MethodsX authors note that the result depends on the negative control and is hard to reproduce. |
Thermo Fisher also advises running ChIPs in replicate and reporting background and standard error.
Which control validates what
The MethodsX paper draws a distinction that is easy to lose. Isotype (IgG) or no-antibody controls show only specificity over background. Positive and negative control genomic regions “validate the accuracy of an assay to a much higher degree”, but they require prior knowledge of at least some true target and non-target sites. An IgG control therefore tells you how much signal is non-specific; it does not tell you that your antibody recognised the intended target.
ChIP versus ChIP-seq, ChIP-exo and ChIP-chip
| Readout | What it adds to the same ChIP enrichment |
|---|---|
| ChIP-qPCR | Quantifies enrichment at loci you chose in advance, using primers; analysed by percent input or fold enrichment as above. |
| ChIP-seq | Sequences the ChIP DNA for a genome-wide view; see the ChIP-seq guide for controls and quality checks. |
| ChIP-chip | Hybridises the ChIP DNA to a microarray; see ChIP-on-chip. |
| ChIP-exo | Adds an exonuclease step that trims the ChIP DNA to a fixed distance from the crosslink site. Rhee and Pugh (Cell 2011) reported single-nucleotide resolution (a standard deviation of about 0.3 bp for Reb1 peak pairs in yeast), tested on yeast Reb1, Gal4, Phd1 and Rap1 and on human CTCF, and described the exonuclease as removing much uncrosslinked nonspecific DNA. |
A related method that does not rely on crosslinking and sonication is covered in CUT&RUN. Protein-protein interaction pull-downs are a different question, covered in co-immunoprecipitation controls and interpretation.
Where it matters for lab decisions
- Choosing the readout: use qPCR when you already know the loci to test; use a sequencing readout when you do not. The enrichment step and its antibody dependence are the same.
- Reporting: state the normalisation method (percent input or fold enrichment), the input fraction, the control used, and the number of replicates, since the published approaches differ in assumptions.
- Biological context: ChIP maps histone marks and factor binding, which is why it appears in work on epigenetics.
Common misunderstandings
Is an IgG control proof that the antibody works?
No. It estimates background only; positive and negative control regions are what test the assay’s accuracy, per the MethodsX paper above.
Is “ChIP assay” a different method from ChIP-qPCR or ChIP-seq?
No. “ChIP assay” usually names the shared immunoprecipitation step; the qPCR, sequencing, array or exonuclease readout is what differs.
Is fold enrichment equivalent to percent input?
No. Fold enrichment depends on a mock control being reproducible; percent input is normalised to the input chromatin, which is why Thermo Fisher’s page prefers it.
FAQ
What does ChIP stand for?
Chromatin immunoprecipitation: antibody-based enrichment of the DNA bound to a chosen protein or histone modification.
What is ChIP-qPCR?
ChIP followed by quantitative PCR at selected loci, with results expressed as percent input or fold enrichment.
What is ChIP-exo?
A ChIP variant that uses an exonuclease to trim the DNA to a fixed distance from the crosslink site, giving near single-nucleotide resolution (Rhee and Pugh, 2011).
What is the ChIP-seq protocol?
ChIP with a sequencing readout. Use a validated protocol from the antibody supplier or published methods; the CASRAI ChIP-seq guide covers controls and quality metrics.
Sources
- Thermo Fisher Scientific, ChIP analysis (percent input and fold enrichment): thermofisher.com, read 2026-11-08.
- A novel method for the normalization of ChIP-qPCR data, MethodsX 2021: PMC8563474, read 2026-11-08.
- Rhee HS, Pugh BF. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 2011;147(6):1408-1419: PMC3243364, checked 2026-11-08.
