Diagenode

Antibodies

Premium Antibodies

The scientific community trusts Diagenode’s antibodies

BLUEPRINT, a part of the IHEC consortium is a large-scale research project which involves 39 leading European universities, research institutes, and industry entrepreneurs. The project aims to generate 100 reference epigenomes of distinct types of haematopoietic cells from healthy individuals and their malignant leukaemic counterparts. To date, BLUEPRINT scientists have isolated cells from 11 bone marrow, 64 venous blood, 46 cord blood, 1 tonsil and 7 cell line samples and have subsequently performed epigenomic analyses  including RNA-seq, DNase-seq and ChIP-seq experiments.

Release composition by assay type

Most of the data (73%) were obtained using ChIP-seq assays. Diagenode was chosen for the development of highly validated antibodies according to strict QC criteria defined by the BLUEPRINT consortium. During this collaboration the following polyclonal antibodies were raised: H2A.Z, H2A.Zac, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me3, H3K9/14ac, H3K9me3, H4K20me3.

These antibodies exhibit the highest level of validation compared to competing antibodies. All are validated in ChIP/ChIP-seq and exibit superior performance for virtually any application. The Premium antibodies passed the most stringent bioinformatic tests (e.g. matching the reference data sets from the Broad Institute). These antibodies are highly specific, sensitive and cost-effective.

  • Read more about our strict and unique quality controls.

    Stringent antibody validation criteria:

    Dot blot

    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.

    Figure 1. Cross reactivity tests using the Diagenode antibody directed against H3K4me3
    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.

    Figure 2. Cross reactivity tests using the Diagenode antibody directed against H3K4me3
    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.

    Western blot

    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
    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

    ChIP-grade

    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%.

    Figure 4. 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. 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-seq grade

    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%.

    Figure 2A

    Figure 2B

    Figure 2C

    Figure 2D

    Figure 5. 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 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.

Products

RFX5-polyclonal-antibody-diagenode

$460.00
The 5-methylcytosine antibody (clone 33D3) is the most published and widely used antibody for DNA methylation analysis. It has been validated for Methylated ...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against histone H2A.Z acetylated at lysines 5, 7 and 11, using a KLH-conjugated synthetic peptide.

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H2A.Z containing the acetylated lysines 5, 7 and 11, using a KLH-conjugated synthetic pept...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the acetylated lysine 27 (H3K27ac), using a KLH-conjugated synthetic peptide.

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 27 (H3K27me3), using a KLH-conjugated synthetic pep...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 36 (H3K36me3), using a KLH-conjugated synthetic pep...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the monomethylated lysine 4 (H3K4me1), using a KLH-conjugated synthetic pept...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 4 (H3K4me3), using a KLH-conjugated synthetic peptide.

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the acetylated lysines 9 and 14 (H3K9/14ac), using a KLH-conjugated syntheti...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 9 (H3K9me3), using a KLH-conjugated synthetic peptide.

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Diagenode is pleased to offer the first recombinant antibody against H3K9me3. It has has been selected using tailored phage-display libraries, and produced i...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic pep...

Histone-Deacetylase-polyclonal-antibody-diagenode

$375.00
Alternative names: HD1, RPD3, RPD3L1, GON-10 Polyclonal antibody raised in rabbit against the C-terminal region of human HDAC1 (Histone deacetylase 1), usin...

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  • Transcription and Chromatin
    EMBL Heidelberg
    Aug 27-Aug 30, 2016
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