|Mapped Reads (HG19)||29,878,289
Table 1. Summary of ChIP-Seq results for EZH2, a low abundant target, performed with Imprint Ultra ChIP kit (CHP2). The results above show a dramatic improvement of the number of sequence reads (23% gain) that map back to the genome of interest in BLASTsearch against the human reference genome.
Figure 3. Sigma’s kit was compared for sensitivity with kits from four other vendors using a low expression transcription factor EZH2. ChIPs were performed with 2 μL of EZH2 Ab (Diagenode, pAb39) and 1 μL of Rabbit IgG (Product No. I5006) with chromatin from 2 million DU145 cells. 2 μL ChIP’ed DNA (out of 30 μL eluate) was analyzed by qPCR using primers for the sonic hedgehog (SHH) gene promoter, and a non-target ZNF333 gene (ZNF333-3’) Sigma’s ChIP kit was the only product that gave unequivocal results, with 45 fold enrichment of target (SHH) and negligible enrichment of non-target (ZNF). Competitors lacked sensitivity and specificity (non-target enrichment to >50% of non-target.
ChIP-qPCR Data Analysis for % Input and Fold Enrichment Calculations (27 Kb Excel File)
Mouse and Rat Primer Sequences
Mouse primers for validating RNAP II ChIPs
|Primer||Sequence||Genomic position (July 2007, mm9)||Amplicon size (bp)|
|Positive Control||m-Actin FP||5' AGCAATAGCCGGAAAGCCAGATCC 3'||chr5:143668924-143669097||174|
|m-Actin RP||5' ACCACTGGGGCTCGCCCTATG 3'|
|Negative Control*||m-Fscn2 FP||5’ CATCCAGGAGCCACTGAAAT 3’||chr11:120222708-120222891||184|
|m-Fscn2 RP||5’ GACATGGACGCTACCTGCTC 3’|
*Barrera, L. et al. 2008. Genome-wide mapping and analysis of active promoters in mouse enbryonic stem cells and adult organs. Genome Res. 18: 46-59. doi:10.1101/gr.6654808
Figure 4. RNAP II ChIP with Mouse Chromatin:
ChIPs were performed with 2.5 mL of RNAP II Ab (Product No. WH0005430M1) and 0.5 mL of Mouse IgG (Product No. I5381) with 250 mL chromatin from NIH 3T3 cells (Active Motif 53021). 2 mL ChIP'ed DNA (out of 30 mL eluate) was analyzed by q-PCR using primers for the Mouse Actin gene promoter, a housekeeping gene and a non-target Fscn2 gene promoter.
Rat primer for validating RNAP II ChIPs
|Primer||Sequence||Genomic position (Rat Nov. 2004)||Amplicon size (bp)|
|Positive Control||r-Actin FP||5' CAGAGAGTCACTCCCCCTTGC 3'||chr12:12046379-12046575||197|
|r-Actin RP||5' CCTCCAGTCACTGGACC 3'|
|Product Name||Product No.|
|Imprint Ultra Chromatin Immunoprecipitation Kit
Imprint Ultra Chromatin Immunoprecipitation Kit without controls
|Imprint Ultra Chromatin Optimizing Kit||CHROP-15RXN|
|Staph-Seq for Chromatin Immunoprecipitation||S6576|
Imprint® ChIP Validated Antibodies
Successful chromatin immunoprecipitation requires an effective, specific, and high quality antibody. Not all antibodies work in ChIP as the antigen epitope may be blocked or altered once cross-linked. Sigma’s Imprint brand of antibodies are validated in ChIP application and lot tested each time for successful chromatin immunoprecipitation experiment. For a complete list of ChIP validated antibodies please click here.