Frequently Asked Questions About Chromatin Immunoprecipitation (ChIP)

For more information about ChIP experiment guides, troubleshooting tips and supplementary protocols, please view our Chromatin Immunoprecipitation (ChIP) page.

Antibodies

1. Should I use a monoclonal or polyclonal antibody?
   Either monoclonal or polyclonal antibodies can work for ChIP. Monoclonals are frequently highly specific, but monoclonals can be sensitive to crosslinking conditions. Over crosslinking may mask the target epitope and careful optimization of crosslinking might be required. Polyclonals are less sensitive to over crosslinking conditions, and may produce better enrichment than comparable monoclonals, but polyclonals are more likely to bind to nonspecific targets.
   
   
2. How much antibody should I use?
   We recommend using 2-10 µg of your ChIP antibody depending on the abundance of your protein target and the affinity of your antibody for the target. More antibody does not always equal stronger signal. It is suggested that to get the best ChIP signal the amount of antibody be titrated.
   
   
3. How can I increase the chances of the selected antibody working in ChIP?
   Test specificity/crossreactivity to identify the epitopes recognized by your antibody. You will also need to test the antibody in multiple immunoassays such as Western blot, immunocytochemistry, and immunoprecipitation, then make sure the antibody produces good fold enrichment of your target DNA in ChIP.
   
   
4. How should I choose a commercial ChIP antibody?
   Choose an antibody that has passed multiple specificity/crossreactivity tests, and validated in ChIP and multiple immunoassays.
   
   
5. What is a good control antibody?
   We recommend using normal IgG from the same species as your ChIP antibody, so if you are using a mouse monoclonal, we recommend normal mouse IgG.
   

Fusion tag

1. What factors should I consider if I want to overexpress a protein with a fusion tag?
   Any system where overexpression is occurring may introduce false-positive interactions. Ideally, if you are analyzing stably expressing fusion-tagged cell lines, we recommend using high and low expressing cell lines, and performing mock IPs from untransfected cell chromatin, for control comparisons.
   
   
2. Do you recommend using tags if I cannot find a suitable ChIP antibody for my study?
   Using a tagged antibody in ChIP is a way to get around antibody unavailability, variability and epitope masking in crosslinked chromatin. It is possible that a tag will interfere with transcription factor function. Tags should be evaluated on a case-by-case basis. Switching tags between N and C termini may be good controls.
   

Cross-link and Beads

1. What is the advantage of using protein A/G bead blend?
   Many antibodies bind to both protein A and G with varying affinity and specificity. Blending protein A and G beads eliminates the need to choose one over the other and to evaluate binding to both types for optimization. In most cases we have seen better fold enrichment and reduced background activity using a protein A/G bead blend compared to using similar quantities of pure protein A or protein G beads.
   
   
2. Is it necessary to quench the crosslinking step?
   Quenching is routinely done with addition of glycine. However, some protocols suggest that washing out the formaldehyde with PBS (when fixing cultured cells) is sufficient to stop crosslinking. This will depend on whether serum is present in the growth media when you are fixing your cells. Glycine quenching is more important if serum proteins are present in the growth media.
   
   
3. Is it necessary to reverse crosslinks and do a solvent extraction of ChIP DNA before agarose gel electrophoresis?
   Crosslink reversal is highly recommended before performing this step. Large complexes may clog the pores of the gel, retard electrophoresis, and produce high background. DNA purification may be optional depending on your sample. Crude DNA can be successfully amplified and stained following proteinase K digestion and crosslink reversal.
   
   
4. Should I consider using non-formaldehyde crosslinkers?
   It may depend on your particular sample. In some cases dual crosslinkers are recommended. UV crosslinking is also done in some cases; however, UV crosslinks are irreversible and will prevent further analysis of your ChIP DNA.
   
   
5. How can you determine if you have over crosslinked your sample?
   If you have not previously studied this target in ChIP, we recommend treating cultured cells with 1% formaldehyde for 10 minutes at RT. If you can’t get enrichment (and expect the protein is present at the location you are looking), then increase crosslinking time to 15 or 20 minutes. If you have overcross linked, in general, you will see the same signal in a location- independent manner. Location-independence means that you observe the same signal at a known binding site versus a known negative locus (such as a 4 kb-distant site).
   
   
6. Is there ever a time when I do not need to cross-link Histones?
   In native ChIP, Histone H3 and Histone H4 do not need to be crosslinked as they are very tightly associated. Histone H2A and Histone H2B are not as tightly associated, but will still work in native ChIP.
   
   
7. The tissue prep procedure calls for making the cross-link solution with formaldehyde in cell culture medium but there is no mention of cell culture medium anywhere else and why would this be used instead of an extraction buffer? What else can be used?
   Cells should be cross-linked (X-linked) in physiological conditions (hence the culture medium) in order to freeze and preserve the DNA-protein interactions in vivo as they happen. X-linked cells are later swollen in hypotonic solution, homogenized to release nuclei and the chromatin is extracted in nuclei lysis buffer.
   

Chromatin Fragmentation

1. Should I perform enzymatic digestion or sonication to shear my chromatin?
   Enzyme digestion may not be as efficient as sonication, and incubating the samples at 37 °C for digestion may result in degradation of epitopes, but digestion may require less optimization than sonication to achieve mono-, di- or trinucleosomes.
   
   
2. What is the best equipment for sonication?
   It depends on your particular sample. It is possible to use any type of sonication equipment, but the sonication process must be optimized for each type of sample.
   
   
3. If using a probe sonicator, where should the probe tip be placed?
   The probe tip should be submerged but not touching the wall of your tube. To prepare chromatin in batch, we recommend using 15 mL conical tubes, with a minimum volume of 0.6 mL (1.2 mL is more consistent), and with a clamp setup to make sure the probe is in the tube near the bottom but not touching the wall. The tube itself should be submerged in an ice bath on an adjustable platform during the sonication process.
   
   
4. I’m having trouble with sonication. Do you have any tips for sonication?
   Keep cells on ice throughout the procedure - even during sonication. Be sure that you don't sonicate for to long (more than 30 seconds could cause sample overheating and denaturation).
   
   Sonication has to be optimized for each cell line and the instrument, we recommend the Diagenode Biodisruptor (water based sonication) for reproducible sonication. A good starting point is 5, 10 and 15 minutes at High “H” setting with 30 seconds “on” and 30 seconds “off” cycle.
   
   Run a gel to check sonication:
   - Use 10 µL sample and add 40 µL H₂O
   - Reverse cross-link by adding 2 µL of 5 M NaCl (Final concentration 0.2 M NaCl)
   - Boil for 15 minutes
   - After returning to room temperature, add 1 µL of 10 mg/ml RNase A at 37 °C for 10 mins
   - Clean and purify DNA with Sigma’s GenElute PCR purification kit
   - Load 1 and 4 µL of sonicated DNA on gel and determine size of smear
   - The sonication condition that gives a smear of DNA sizes from 200 bp to 1 kb with a peak around 500 bp (2-3 nucleosomes) should be used for ChIP reactions.
   
   
5. Why do you have to shear the DNA down less than 1000 base pairs (to about three nucleosomes ~400-500bp)?
   To insure good resolution for ChIP. If your average fragment size is greater than 1000 bp, you could be pulling down DNA that contains your target sequence for PCR but the protein of interest may be over 700 nucleotides distant from your target.
   

Cell #s, buffers, nuclei isolation and others

1. Will subjecting my chromatin to freeze-thaw cycles affect my ChIP results?
   We don’t recommend freeze-thaw cycles when handling chromatin samples, but in some cases, this has not affected the ChIP result. This may depend on the buffer in which the chromatin is stored. In some protocols, glycerol is included in the buffer to stabilize epitopes through freeze-thaw cycles.
   
   
2. Why are high volumes of ChIP dilution buffer required before IP? Are different buffers preferred for different beads?
   Ten-fold dilution of chromatin is necessary when the lysis buffer used in chromatin fragmentation contains high concentrations of SDS (that is 1% in some cases). Diluting the chromatin ensures that the IP beads and antibody won’t be denatured or affected by the presence of the detergent. We use similar buffers for both agarose beads and magnetic beads in our ChIP kits.
   
   
3. Is a nuclei isolation step required?
   Isolating nuclei prior to extraction of chromatin can reduce background by eliminating cytoplasmic proteins.
   
   
4. How can I tell if I have isolated nuclei from my cells?
   You can view the samples on a phase contrast microscope to determine if the nuclei have been released from cells.
   
   
5. How many cells should be used for targets such as accessory factors that are indirectly associated with DNA?
   Ten million cells is recommended.

6. Can the number of cells be increased? What is the maximum and minimum number of cells that can be used with Imprint^® Chromatin Immunoprecipitation Kit?
   The range of cells that can be used with this kit is 0.1-1 million/well/ChIP sample, using more than 1 million cells per well will increase the non-specific binding and reduce the specificity of the ChIP reaction. If highwe yield of ChIP DNA is desired DNA could be pooled from multiple individual ChIP reactions for downstream processing (labeling/ hybridization) or DNA could be amplified using the WGA 2 kit for ChIP-chip analysis.
   
   
7. What is the GAPDH primers efficiency for doing qPCR?
   The GAPDH primers efficiency for doing qPCR is 95 %.
   
   
8. Is the Imprint^® Chromatin Immunoprecipitation Kit compatible with lysing enzymes if sonication does not work?
   Yes, sheared chromatin can also be made by enzymatic (Micrococcal nuclease, MNase) digestion (reagents/protocol not provided in kit). The amount and duration of the MNase treatment will have to be optimized by the user depending on the cell line.
   
   
9. What is the function of nuclear preparation buffer? When do you add the protease inhibitor?
   The nuclear preparation buffer is a hypotonic salt solution that serves to swell the cells and facilitate the release of nuclei during the subsequent homogenization step. The protease inhibitor cocktail should be added just before use.
   
   
10. Can Imprint® Chromatin Immunoprecipitation Kit be used with plants? Reptilian cells?
    Yes, the kit is compatible with sonicated chromatin prepared from plants or reptilian cells provided the user has optimized conditions of cross-linking and preparation of appropriately sized sonicated chromatin.
    
    
11. How should I perform ChIP with tissues?
    In general, chromatin can be prepared from fresh or frozen tissue, and fixed in 1% formaldehyde-containing solutions for 15 minutes, but there are many variations on how this can be done and how tissues are disaggregated or homogenized prior to chromatin isolation. You may also try the Magna ChIP G Tissue Kit (17-20000) for an easier approach to processing your tissue samples for ChIP. There are also protocols available for tissue ChIP from laboratories such as the Farnham lab at UC Davis, as well as several published methods for brain tissue.
    
    
12. How many ChIP reactions can I perform at once?
    Commercial methods now exist to enable you to perform up to 96 ChIP reactions at once in a 96-well plate. For high throughput studies, we suggest our Magna ChIP™ HT96 ChIP Kit (17-10077) or the EZ-Magna ChIP™ HT96 Kit (17-10078) with included controls.

Data Analysis

1. Should I perform gel analysis or quantitative real-time PCR (qPCR)?
   We recommend qPCR because it eliminates the need to optimize cycle number to ensure you are in the logarithmic amplification phase of your assay. Enrichment may be lower with transcription factors, but qPCR allows you to be more quantitative rather than qualitative.
   
   
2. If I wanted to quantitate my immunoprecipitated DNA, how would I do so?
   DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.⁰C (+/- 2.0). You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment to optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin.
   
   Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required.
   
   Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93.
   
   
3. How do you measure percent enrichment?
   One approach is to run concurrent PCR reactions of replicate serial dilutions of the input DNA alongside your ChIP DNA and antibody control samples. The Ct values obtained can be used to plot a standard curve and the test samples can be fit to this curve to determine percent enrichment. (see section on qPCR and data analysis).
   
   
4. How do I report error (intra-assay or inter- assay variation) for qPCR results?
   Conduct all qPCR in replicates, and calculate the mean Ct and standard error. Then, propagate that standard error throughout your calculations of enrichment using an error propagation calculator, such as https://www.eoas.ubc.ca/courses/eosc252/error-propagation-calculator-fj.htm
   
   
5. Is it advisable to normalize the test sample to just IgG in the relative Ct method?
   The standard practice for comparative analyses is to use mock IP, ChIP IP, and input on both a target amplicon and a reference amplicon. Using just mock IP (IgG) Ct values as a normalizer without the additional samples required is not advisable.
   
   
6. What is an acceptable % input range for the normal IgG control antibody?
   The IgG pulldown can be quite variable and qPCR-assay dependent. The same mock IgG sample can have different percent input results in one location of the genome vs. another region based on sequence composition of the assay design. The signal may be the result of nonspecific binding of nucleic acid to tube, to beads, to antibodies. ChIP is relative so it is best not to attempt to conform to a specific percent of input value, but ideally, the IgG value should have Ct values that are nearest to the most dilute sample in your standard curve. Again, ChIP is relative so if your ChIP signal is higher than your IgG signal (within limits of variation in the assay), you have a positive ChIP result.
   
   
7. What is considered a good fold enrichment of my ChIP DNA?
   This will depend on your protein target. Abundant targets will likely produce high enrichments relative to low abundance targets. Some labs set a minimum fold enrichment of 5-fold over an IgG control as a minimum threshold for a successful experiment. However, it is best to compare your results to published results rather than conform to a set value.
   
   
8. How can I test my qPCR primers?
   You can test your primers by running a qPCR reaction with serial dilutions of a suitable sample of known concentration. If your primers are working optimally, and all other conditions of the reaction have been satisfied, you should observe an approximate doubling of amplicon in each linear phase of the cycle (see section on qPCR and data analysis).
   
   
9. Should I perform a singleplex or multiplex qPCR assay?
   A multiplex experiment may be more suitable when testing multiple samples but will require more complex detection methods such as fluorophore-tagged probes that can easily produce distinct fluorescent signals for each target DNA in your reaction tube. You will therefore need to perform optimizations for each of your primer-probe set and you may incur additional costs. A singleplex experiment is fairly easy to design and multiple samples can be run in parallel with cost-effective DNA-binding dyes such as SYBR^® Green and appropriately designed primers (see section on qPCR and data analysis).
   
   
10. How can I distinguish nucleic acid contaminants from nonspecific binding of SYBR^® Green?
    You can perform a melt curve analysis in which you manipulate the thermal conditions of the qPCR process to allow your amplicons to melt. You should observe only one peak in the melt curve for your target amplicon. Additional peaks may indicate the presence of primer dimers or contaminants.