No/Low Target DNA Enrichment

No target band detected by qPCR/sequencing, or enrichment fold ＜ 2

1. Excessive/insufficient formaldehyde crosslinking, affecting protein-DNA binding or sonication;

2. Poor sonication conditions, resulting in over-long (>1000bp) or over-short DNA fragments;

3. Low antibody specificity, failing to bind target protein effectively;

4. Over-washing leading to dissociation of protein-DNA complexes

1. Optimize crosslinking (1% formaldehyde, 5-10min at RT, terminated by glycine);

1. Adjust sonication power/time to obtain 200-500bp DNA fragments;

2. Use ChIP-grade specific antibodies and set positive control antibodies;

4. Reduce washing cycles (3-4 times) and lower wash buffer salt concentration

High Background (Non-specific DNA Enrichment)

High enrichment fold of negative control (IgG), poor specificity

1. Non-specific binding of antibody to chromatin;

2. Incomplete sonication, large DNA fragments prone to non-specific adsorption;

3. Inadequate blocking, magnetic/agarose beads adsorbing heteroprotein-DNA;

4. Incomplete washing with residual unbound chromatin

1. Set IgG isotype control and select high-specificity ChIP-grade antibodies;

2. Optimize sonication to ensure uniform DNA fragmentation;

3. Block beads with salmon sperm DNA + BSA for 1h;

4. Increase washing cycles (4-5 times) and use wash buffer containing 0.1% SDS

Low DNA Recovery Rate

Low eluted DNA concentration, unable to meet subsequent detection

1. Insufficient starting cell number (<1×10⁶ cells);

2. Excessive sonication causing DNA fragmentation into non-recoverable small fragments;

3. Mild elution conditions, incomplete dissociation of protein-DNA complexes;

4. Severe DNA loss during purification

1. Increase starting cell number (1×10⁷~1×10⁸ cells);

2. Reduce sonication power to avoid over-fragmentation;

3. Optimize elution (50mM Tris-HCl pH8.0 + 10mM EDTA + 1% SDS, incubate at 65℃ for 15min);

4. Use high-efficiency DNA purification kits and reduce purification steps

Dissociation of Protein-DNA Complexes

Target protein or DNA undetectable after immunoprecipitation

1. Insufficient crosslinking, weak-binding complexes prone to dissociation;

2. Excessively high incubation temperature disrupting protein-DNA interaction;

3. High detergent concentration in wash buffer

1. Extend crosslinking time (for weak interactions) or use dual crosslinkers;

3. Perform all incubations (antibody binding, washing, precipitation) at 4℃;

3. Reduce SDS concentration in wash buffer to below 0.1%

No PCR Amplification/Low Amplification Efficiency

No PCR product detected from eluted DNA

1. DNA degradation (no nuclease inhibitors added during operation);

2. Residual SDS/proteinase K in eluted DNA inhibiting Taq enzyme activity;

3. Improper PCR primer design (excessive span or low specificity)

1. Add RNase/DNase inhibitors throughout the process to prevent DNA degradation;

3. Purify eluted DNA to remove residual inhibitors;

3. Design specific primers targeting 200-500bp fragments and set positive control primers

Poor Experimental Reproducibility

Large variation in enrichment fold between batches

1. Inconsistent cell culture conditions;

2. Unstable sonication parameters;

3. Antibody batch differences;

4. Poor control of incubation time/temperature

1. Standardize cell culture and use cells of the same passage;

2. Fix sonication power, time and cycles;

3. Use antibodies from the same ChIP-grade batch and store in aliquots;

4. Strictly control incubation at 4℃ overnight (antibody binding)