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ChIP-seq Validated Antibodies

A successful ChIP-seq experiment requires an antibody that recognizes the correct target protein in all sequence contexts across the entire genome. Good antibody performance in ChIP-qPCR does not necessarily mean the antibody will perform well for ChIP-seq, because ChIP-seq requires more extensive capture of the target protein across a large number of gene loci. To provide antibodies that are proven effective for ChIP-seq, CST validates recombinant rabbit monoclonal antibodies for ChIP-seq using the SimpleChIP® Enzymatic and SimpleChIP® Sonication Chromatin IP protocols followed by next-generation sequencing (NGS).

ChIP-seq Antibody Validation Steps

  • All ChIP-seq validated antibodies are first subjected to the ChIP-qPCR validation protocol.
  • Antibody sensitivity for ChIP-seq is then confirmed by analyzing the signal:noise ratio of target enrichment across the genome in antibody:input control comparisons. The antibody must provide an acceptable minimum number of defined enrichment peaks and a minimum signal:noise threshold compared to input chromatin.
  • For sequence-specific DNA-binding transcription factors, antibody specificity is determined by performing motif analysis of enriched chromatin fragments.
  • Antibody specificity is further determined by comparing enrichment across the genome using multiple antibodies against distinct target protein epitopes.
  • Antibody specificity is confirmed using antibodies against different subunits of a multiprotein complex.
  • Antibody specificity is further confirmed by comparing enrichment across the genome to published ChIP-seq data (ie, ENCODE) using additional antibodies for a given target protein.

Signal:Noise (feature #2569)

Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP® Enzymatic Plus Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows a specific binding of TCF4/TCF7L2 across c-MYC, a known target gene of TCF4/TCF7L2 (upper track), compared to Input control (lower track). The average signal:noise ratio across the c-MYC gene is 21.58.

Chromatin immunoprecipitations

Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP® Enzymatic Plus Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. As shown in the figure, the average signal:noise ratio across the whole genome for this experiment, comparing the TCF4/TCF7L2 enrichment (blue line) to the Input control (red line), is 4.44.

Antibody Specificity for Transcription Factors

Chromatin immunoprecipitations were performed with cross-linked chromatin from A549 cells cultured in media with 5% charcoal-stripped FBS for 3 d and then treated with 100 nM dexamethasone for 1 hour and Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows the specific binding of GR across SLC19A2, a known target gene of GR (upper track), compared to the input control (lower track). Forty-one percent (41%) of the GR antibody-enriched DNA fragments contain the known glucocorticoid response element binding motif GNACANNNTGTNC.

Antibody Specificity for Protein Complexes

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d, followed by treatment with β-estradiol (10 nM, 45 min) and either SMARCC1/BAF155 (D7F8S) Rabbit mAb #11956, SMARCB1/BAF47 (D8M1X) Rabbit mAb #91735, or SS18 (D6I4Z) Rabbit mAb #21792, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. SMARCC1/BAF155, SMARCB1/BAF47, and SS18 are all subunits of SWI/SNF complex. As expected, all three protein subunits of the SWI/SNF complex show overlapping binding across pS2/TFF1, a known target gene of the SWI/SNF complex.