Stringent antibody validation criteria:
The specificity of antibodies against modified proteins (e.g. histones) is tested by dot blot. The signal obtained with the specific peptide should be >90% of the total signal on the blot for the highest peptide concentration.
To test the cross-reactivity of the Diagenode antibody against H3K4me3 (cat. No. C15410003), a dot blot analysis was performed with peptides containing other histone modifications and the unmodified H3K4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:10,000. Figure 1 shows a high specificity of the antibody for the modification of interest.
Modified histones peptide array
The specificity of antibodies against modified histones is further tested on peptide arrays. These arrays contain 384 different peptides in duplicate with different combinations of H3, H4, H2A and H2B modifications. A specificity factor >30 and at least 5x higher than for any other modification is a required for the validation of Premium antibodies.
The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:2,000. Figure 2 shows a high specificity for the peptides containing the H3K4me3 modification.
The overall specificity of the antibodies is tested in Western blot performed on whole cell extracts, histone extracts and recombinant histones H2A, H2B, H3 and H4. The following criteria are applied for Premium antibodies:
- A specific signal >80% of the total signal in the lane containing the whole cell extracts
- Signal of other histones <10% of the total signal in the lane containing the histone extracts
- Signal with any of the recombinant histones <10% of the specific signal in the lane with the histone extracts
Western blot was performed on whole cell (40 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3K4me3 (Cat. No. C15410003). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left
ChIP is performed according to our standardized protocol. qPCR is performed using at least 2 positive and 2 negative control targets. To pass the ChIP QC, the antibody has to show the expected profile with a +/- ratio >5. The recovery of the positive control targets should be >1%.
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K4me3 (Cat. No. C15410003) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010022), using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes
ChIP for ChIP-seq is performed on sheared chromatin with the iDeal ChIP-seq kit. Extensive bioinformatic analysis is applied: peak detection with SICER and %RIP >40; peak comparison with published data (Broad Institute) and overlap of the top 40 most significant peaks >90%.
ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 μg of the Diagenode antibody against H3K4me3 (Cat. No. C15410003) as described above. The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 5 shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure 5A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 5C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.