Chromatin Immunoprecipitation Troubleshooting Guide

Select the ChIP Problem you are Encountering Concentration is too Low Fragments are too Large Fragments are too Small No or Little Product in the PCR Reactions No Product in Positive Control No Product in Experimental Antibody-IP PCR Reaction

A. Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table below provides ranges for expected total yield of chromatin and expected DNA concentration from 25 mg of tissue or 4 x 10⁶ HeLa cells, as determined in Section IV of the protocols.

• In the SimpleChIP^® Enzymatic protocol, disaggregation using a BD^™ Medimachine system (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, tissue disaggregation using the Mediamachine typically gave higher IP efficiencies compared to disaggregation using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension.
• In the SimpleChIP^® Sonication protocol, a Dounce homogenizer is recommended for all tissue types.

For optimal ChIP results, we recommend using 5 to 10 µg of cross-linked and fragmented chromatin per IP; therefore, some tissues may require harvesting more than 25 mg per each IP.

NT = not tested

B. Optimization of Chromatin Fragmentation

In the SimpleChIP^® Enzymatic protocol, optimal conditions for the digestion of cross-linked chromatin DNA to 150–900 bp fragments is highly dependent on the ratio of micrococcal nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

1. Prepare cross-linked nuclei from 125 mg of tissue or 2 x 10⁷ cells (equivalent of 5 IP preps), as described in Protocol Sections I, II, and III. Stop after Step 2 of Protocol Section III and proceed as described below.
2. Transfer 100 μl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
3. Add 3 μl micrococcal nuclease stock to 27 μl of 1X Buffer B + DTT (1:10 dilution of enzyme).
4. To each of the 5 tubes in Step 2, add 0 μl, 2.5 μl, 5 μl, 7.5 μl, or 10 μl of the diluted micrococcal nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
5. Stop each digest by adding 10 μl of 0.5 M EDTA and placing tubes on ice.
6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
7. Resuspend nuclear pellet in 200 μl of 1X ChIP buffer + PIC. Incubate on ice for 10 min.
8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei on a light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20 sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
10. Transfer 50 μl of each of the sonicated lysates to new microfuge tubes.
11. To each 50 μl sample, add 100 μl nuclease-free water, 6 μl 5 M NaCl and 2 μl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
12. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 hr.
13. Remove 20 μl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
14. Observe which of the digestion conditions produces DNA in the desired range of 150–900 base pairs (1–6 nucleosomes). The volume of diluted micrococcal nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of micrococcal nuclease stock that should be added to one IP preparation (25 mg of disaggregated tissue cells or 4 x 106 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 μl of diluted micrococcal nuclease produces DNA fragments of 150–900 bp in this protocol, then 0.5 μl of stock micrococcal nuclease should be added to one IP preparation during the digestion of chromatin in Section III.
15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of micrococcal nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

In the SimpleChIP^® sonication protocol, optimal conditions for the fragmentation of cross-linked chromatin DNA are highly dependent on the number of cells, volume of sample, length of sonication, and sonicator power setting used. For each sonication sample, we recommend using 100–150 mg of tissue or 1 x 10⁷–2 x 10⁷ cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Below is a protocol for determining the optimal sonication conditions for a specific tissue or cell type.Resuspend nuclear pellet in 200 μl of 1X ChIP buffer + PIC. Incubate on ice for 10 min.

1. Prepare cross-linked nuclei from 100–150 mg of tissue or 1 x 10⁷–2 x 10⁷ cells, as described in Sections I, II, and III. Stop after Step 4 of Section III and proceed as described below.
2. Fragment chromatin by sonication. Optimal sonication conditions can be determined for a given sonicator by varying the number of rounds or duration of sonication at a given power setting (see Step 5 in Section III for optimal power setting using a Branson Digital Sonifier 250 probe sonicator). To determine optimal sonication conditions, set up a sonication time-course experiment and remove 50 μl samples of chromatin after a given round or duration of sonication. For example, take chromatin samples after each 1 to 2 min of sonication.
3. Clarify chromatin samples by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
4. Transfer supernatants to new microfuge tubes and add 100 μl nuclease-free water, 6 μl 5 M NaCl #7010, and 2 μl RNAse A #7013. Vortex to mix and incubate samples at 37°C for 30 min.
5. To each RNase A-digested sample, add 2 μl Proteinase K #10012. Vortex to mix and incubate samples at 65°C for 2 hr.
6. Remove 20 μl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
7. Choose the sonication conditions that generate optimal DNA fragment size (see note below) and use for chromatin preparation in Step 5 of Section III. If optimal sonication conditions are not achieved, increase or decrease the power setting of the sonicator and repeat the sonication time course.

NOTE: Optimal sonication conditions can vary with different sample types and fixation times. Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments. Over-sonication, indicated by >80% of total DNA fragments being shorter than 500 bp, can result in excessive damage to the chromatin and lower immunoprecipitation efficiency.

• For sonication of cells fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 90% of total DNA fragments less than 1 kb. Increasing the fixation time to 30 min will reduce fragmentation, generating a DNA smear with approximately 60% of total DNA fragments less than 1 kb.
• For sonication of tissues fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 60% of total DNA fragments less than 1 kb. Increasing the fixation time to 30 min will reduce fragmentation, generating a DNA smear with approximately 30% of total DNA fragments less than 1 kb.

C. Troubleshooting Table

Problem	Possible Causes	Recommendation
Problem	Possible Causes	Recommendation
1. Concentration of the fragmented chromatin is too low.	Not enough cells or tissue were used for the chromatin preparation or cell/tissue lysis was incomplete.	If DNA concentration of the chromatin preparation is close to 50 μg/ml, add additional chromatin to each IP to give at least 5 μg/IP and continue with protocol.
		Count a separate plate of cells before cross-linking to determine an accurate cell number.
		Enzymatic: visualize cell nuclei under microscope before and after sonication to confirm complete lysis of nuclei.
2. Chromatin is under-fragmented and fragments are too large. Large chromatin fragments can lead to increased background and lower resolution.	Cells may have been over-crosslinked and/or too much input material (cells/tissue) was processed.	Shorten the crosslinking time within 10–30 minute range and/or reduce the amount of cell/tissues per sonication.
		Enzymatic: increase the amount of Micrococcal nuclease to the chromatin digestion or perform a time course for enzymatic digestion.
		Sonication: conduct a sonication time course.
3. Chromatin is over-fragmented. Digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes.	Enzymatic: Not enough cells or too much Micrococcal nuclease added to digestion.	Enzymatic: weigh tissue or count a separate plate of cells prior to cross-kinking to determine accurate cell number. Add more tissue or cells, or less Micrococal nuclease to the chromatin digest.
	Sonication: Conditions are too harsh.	Sonication: conduct a sonication time course to find a minimum output/duration to achieve appropriate sonication.
4. No product or very little product in the input PCR reactions.	Not enough DNA added to the PCR reaction or conditions are not optimal.	Add more DNA to the PCR reaction or increase the number of amplification cycles. Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and fragmented chromatin.
	PCR amplified region may span nucleosome-free region.	Design a different primer set and decrease length of amplicon to less than 150 bp.
	Not enough chromatin added to the IP or chromatin is over-fragmented.	For optimal ChIP results add 5–10 μg chromatin per IP. See recommendations for problems 1 and 3 above.
5. No product in the positive control Histone H3-IP RPL30 PCR reaction.	Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short.	Be sure to add 5–10 μg of chromatin and 10 μl of antibody to each IP reaction and incubate with antibody over-night and an additional 2 hr after adding Protein G beads.
	Incomplete elution of chromatin from Protein G beads.	Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.
6. Quantity of product in the negative control Rabbit IgG-IP and positive control Histone H3-IP PCR reactions is equivalent.	Too much or not enough chromatin added to the IP reaction. Alternatively, too much antibody added to the IP reaction.	Add no more than 15 μg of chromatin and 10 μl of histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 μl per IP.
	Too much DNA added to the PCR reaction or too many cycles of amplification.	Add less DNA to the PCR reaction or decrease the number of PCR cycles. For accurate quantitation, it is critical that PCR products are analyzed within the linear amplification phase of PCR.
7. No product in the Experimental Antibody-IP PCR reaction.	Not enough DNA added to the PCR reaction.	Add more DNA to the PCR reaction or increase the number of amplification cycles.
	Not enough antibody added to the IP reaction.	Typically a range of 1–5 μg of antibody is added to the IP reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP.
	Antibody does not work for IP.	Choose an alternate, ChIP-validated antibody.

posted March 2008

revised May 2017

Support: 877-678-8324 support@cellsignal.com • Orders: 877-616-2355 orders@cellsignal.com • Web: www.cellsignal.com