Troubleshooting of Chromatin Immunoprecipitation (ChIP)

The following troubleshooting guide is intended to explain causes and possible solutions for common problems observed in chromatin immunoprecipitation (ChIP) application. Here you can find some common issues and solutions you can use to fix them. Though the tips provided here cover many different problems you may encounter in ChIP, we hope that you will find the information beneficial to you and useful as a reference guide. If you ever need more assistance with your experiments, please contact us for additional help. Protocol of Chromatin Immunoprecipitation (ChIP) is available for you.

Contents

High Background in Non-specific Antibody Control
Weak or No Signal
Low Resolution with High Background Across Large Regions
PCR Amplification Problems
Low Recovery of DNA
Cell Lysis
Chromatin Shearing
Negative ChIP Control(s)

High Background in Non-specific Antibody Control

Possible Cause	Recommended Solution
Non-specific binding to beads	Add additional washes. Add a blocking agent to the beads. Add a pre-clearing step by incubating sonicated chromatin with protein A/G beads for 1 h prior to immunoprecipitation.
Excessive antibody resulting in binding to non-targets	Optimize the concentration of the antibody.
Contaminated wash buffers	Change buffers.
Incomplete fragmentation of chromatin	Optimize the fragmentation process to acquire chromatin lengths between 200-1000 bp. Separately optimize fragmentation for each cell or tissue type. Use siliconized or low retention tubes.

Weak or No Signal

Possible Cause	Recommended Solution
Cells not efficiently lysed	Use appropriate buffer.
Not enough starting material	ChIP normally requires a large input with at least 25 µg chromatin (3-4 million cells) per IP condition.
Chromatin fragment size may be too small	Run on a gel to ensure correct size. Repeat fragmentation optimization if necessary.
Not enough antibody	3-5 µg is usually sufficient, but up to 10 µg may be required if no signal is observed.
Monoclonal antibodies may not be suitable	Try a polyclonal antibody or ChIP grade/approved monoclonal antibody.
Wash buffer is too stringent	NaCl in buffer should not exceed 500 mM. Optimize wash buffer.
Wrong affinity beads	Make sure antibody species and immunoglobulin bind to chosen beads or use a protein A/G mix.

Low Resolution with High Background Across Large Regions

Possible Cause	Recommended Solution
DNA fragment size may be too large	Fragment sizes should be less than 1 kb, but ideally 200-1000bp. The best resolution can be achieved with micrococcal nuclease (MNase) digestion to single nucleosome level of 175 bp. Run on a gel and further optimize chromatin fragmentation steps if necessary.

PCR Amplification Problems

Possible Cause	Recommended Solution
qPCR solution may be contaminated	Prepare new solutions from stock.
Primers may not be working	Include input DNA control.
“No DNA” PCR reaction is showing signal	Use pipettes dedicated to PCR, and UV-irradiate pipettes prior to setting up PCR. Perform ChIP, DNA purification, and PCR reaction setup in three separate rooms using dedicated pipettes or set up reactions in a hood. Avoid using bottled or otherwise prepackaged water. Do not open tubes containing amplified PCR products anywhere near the location of future ChIP or qPCR experiments.
No amplification with input DNA	Ensure that you are using an antibody that has been validated in ChIP. Increase the incubation time of the antibody. Increase the antibody amount. Increase your cell number especially if you are attempting to detect a low abundance target.
No amplification of product	Not enough antibody. Verify that your primers are properly designed and that your thermal cycler protocol is agreeable with your Taq master mix. Use more template DNA.

Low Recovery of DNA

Possible Cause	Recommended Solution
Ineffective or low affinity ChIP antibody	Ensure that you are using an antibody that has been validated in ChIP. Increase the incubation time of the antibody.
Insufficient ChIP antibody	The amount of antibody required may depend on the relative abundance of your target protein and the affinity of the antibody for the target.
Insufficient starting sample	Before crosslinking, prepare a separate plate to determine cell number. Re-evaluate your cell number per reaction. Increase your cell number if you are attempting to detect a low abundance target.
Incomplete cell lysis and ineffective fragmentation	Optimize these steps by varying parameters and assessing their effects on chromatin recovery. Use mechanical force such as glass beads to improve cell lysis. Optimize the fragmentation steps and avoid foaming.
Over crosslinking	Optimize crosslinking steps: the final concentration of formaldehyde should be 1%. Determine the most effective crosslinking time before proceeding with the experiment.
Under crosslinking	Use an X-ChIP protocol to stabilize the associations of your target protein with DNA. Increase crosslinking time.

Cell Lysis

Possible Cause	Recommended Solution
Temperature is critical	Perform cell lysis at 4°C or on ice. Use ice-cold buffers.
Protein degradation during lysis	Add the protease inhibitors to the lysis buffer immediately before use.

Chromatin Shearing

Shearing conditions for each cell type must be optimized from cell collection, fixation to shearing method (settings of the sonicator apparatus).
Not to start with a too high concentration of cells (no more than 1.5 x 10⁷ cells/mL).
Keep samples at 4°C or on ice.

Negative ChIP Control(s)

Use the non-immune IgG fraction from the same species the antibodies were produced in.
Use one antibody in ChIP and the same antibody that is blocked with specific peptide. Pre-incubate the antibody with saturating amounts of its epitope specific peptide for about 30 min at RT before use in the IP incubation mix. Use the blocked antibody as a negative control in parallel with the unblocked antibody.