Comparison of time required for protocol completion from fixation through purification using different Chromatin Immunoprecipitation kits. Each ChIP experiment was carried out using each manufacturers recommended protocol and total required times were compared.

One day is sufficient to acquire ChIP enriched DNA using Sigma′s Imprint ChIP protocol. ChIP with antibodies against mouse IgG, H3K9ac, RNAP and H3K27me3 was completed on 10,000 and 100,000 SW480 cells per reaction. A fraction (1%) of the sonicated chromatin was set aside as ′input′ DNA before the antibody affinity manipulations. DNA was purified following crosslink reversal. The resulting enriched DNA was probed for GAPDH using specific primers by qPCR. Percent input was calculated by 100 × 2^(Ctadjusted Input – CtEnriched). Input DNA Ct was adjusted from 1% to 100% equivalent by subtracting 6.644 Cts or Log2 100.

Figure 3. A highly sensitive ChIP assay. HeLa cells were counted, fixed, and immunoprecipitated according to the Imprint CHP1 bulletin with optional High Sensitivity method. ChIP assays were performed using Anti-H3K9ac (H9286) and kit provided antibodies against Human RNA Polymerase II and non-specific Mouse IgG. To evaluate ChIP enrichment, SYBR qPCR was conducted targeting the GAPDH promoter region (highly expressed house-keeping gene). Percent Input describes the amount of DNA with and without antibody selection. For these antibodies, apparent enrichment increased with fewer cells per reaction from diminished non-specific pull-down.

Figure 4. A highly sensitive ChIP assay. HeLa cells were counted, fixed, and immunoprecipitated according to the Imprint CHP1 bulletin with optional High Sensitivity method. ChIP assays were performed using Anti-H3K9ac (H9286) and kit provided antibodies against Human RNA Polymerase II and non-specific Mouse IgG. To evaluate ChIP enrichment, SYBR qPCR was conducted targeting the GAPDH promoter region (highly expressed house-keeping gene). Percent Input describes the amount of DNA with and without antibody selection. For these antibodies, apparent enrichment increased with fewer cells per reaction from diminished non-specific pull-down.