ChIP Protocol

critical

No Product in Histone H3 Positive Control IP

Positive control Histone H3-IP with RPL30 primer set produces no PCR product, indicating fundamental problems with IP procedure or elution.

💡 4 · ✓ 4

critical

No or Minimal PCR Product in Input Control

Input chromatin PCR reactions produce no product or very little product, indicating problems with DNA quantity, PCR conditions, or primer design.

💡 5 · ✓ 5

critical

Antibody Lacks Specificity or ChIP Validation

ChIP-seq generates high background signal across the genome with poor enrichment at expected binding sites. Signal-to-noise ratio is low, and peaks are difficult to distinguish from background.

💡 4 · ✓ 5

severe

Low X-ChIP Signal from Excessive Formaldehyde Cross-Linking

Low signal specifically in X-ChIP (cross-linked ChIP) experiments. Antibody appears unable to bind target epitopes despite proper antibody concentration and chromatin quality.

💡 4 · ✓ 4

severe

Low Fragmented Chromatin Concentration

DNA concentration of fragmented chromatin preparation is below expected ranges (e.g., <100 µg/ml for HeLa cells, <20 µg/ml for brain tissue). Insufficient material for recommended 5-10 µg chromatin per IP reaction.

💡 4 · ✓ 5

severe

Chromatin Over-Fragmentation to Mono-Nucleosome Length

More than 80% of DNA fragments are shorter than 500 bp, resulting in diminished PCR signal especially for amplicons >150 bp, and potential disruption of chromatin integrity and antibody epitopes.

💡 4 · ✓ 4

moderate

Low ChIP Signal from Ineffective Cell Lysis

Overall low signal with visible cell clumps or debris in lysate. Chromatin yield is lower than expected based on starting cell number.

💡 4 · ✓ 4

moderate

Low ChIP Signal from Insufficient Starting Material or Antibody

Weak or barely detectable signal across all samples including positive controls. Signal intensity is uniformly low rather than selectively absent at specific regions.

💡 4 · ✓ 4