Quality matters - That’s why we validate antibodies at Diagenode

Obtaining highly sensitive and highly specific antibodies is challenging for researchers. Many antibodies that are used in experimental assays have limited validation behind them, and the data given by the provider are not always lot-specific. Consequently, researchers must often independently validate antibodies, a time-consuming and costly process.

Diagenode has addressed these issues and provides:

• A rigorous QC process using stringent criteria for antibody validation
• Accessible validation data presented in our data sheets and website, obtained with current lots of the antibodies
• Validation standardization - Every new lot is tested using the same stringent criteria and compared to the previous lot before adding to the catalog.

1. Peptides are designed to evoke a maximal immune response and coupled to KLH

2. Up to 20 rabbits arre immunized. Each rabbit is injected 6 times. Four bleeds are taken from each rabbit.

3. All bleeds are tested by ELISA and WB. Antibodies against modified histones are also tested for specificity against different peptides with related modifications by dot blot.

4. Only bleeds which show a single band of the expected size in WB or at least 90% specificity in dot blot are selected for purification

5. The purified antibodies are again tested in ELISA, dot blot and WB.

6. Purifications that pass this QC are pooled together giving a final lot, which is again tested.

7. The specificity of the final lot is further demonstrated by peptide or protein arrays. Diagenode also has introduced WB combined with siRNA blocking of the target expression as an additional validation procedure. Finally, the antibodies are validated in ChIP-seq

Figure 1. Cross reactivity tests using the Diagenode antibody directed against H3K4me1
To test the cross reactivity of the Diagenode antibody against H3K4me1 (Cat. No. C15410194), 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 1:5,000. Figure 1 shows a high specificity of the antibody for the modification of interest.

Figure 2. Cross reactivity tests using the Diagenode antibody directed against H3K4me3
The specificity of the antibody was 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.

Figure 3. Western blot analysis using the Diagenode antibody directed against H3K4me3
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

Figure 4. Western blot analysis using the Diagenode monoclonal antibody directed against HDAC1
Whole cell extracts (40 μg) from HeLa cells transfected with HDAC1 siRNA (lane 2) and from an untransfected control (lane 1) were analysed by Western blot using the Diagenode antibody against HDAC1 (Cat. No. C15100144) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right (expected size: 55 kDa); the marker (in kDa) is shown on the left.

Figure 5. IF analysis using the Diagenode antibody directed against H3K4me1
IF using the Diagenode H3K4me1 antibody (Cat. no. 15410194), diluted 1:200 after incubation with 4 different blocking peptides (H3K4un, H3K4me1, H3K4me2 and H3K4me3). The image shows the merged staining with the antibody (green) and with DAPI (blue). The green signal only disappears after blocking with the H3K4me1 peptide.

Figure 6. ChIP results obtained with the Diagenode antibody directed against H3K4me3
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. The figure 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.

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Figure 8. ChIP-seq results obtained with the Diagenode antibody directed against H3K4me3
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 7 shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure 8A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 8C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.