Polyclonal antibody raised in rabbit against histone H3, trimethylated at lysine 36 (H3K36me3), using a KLH–conjugated synthetic peptide

ChIP-seq results obtained with the antibody directed against H3K36me3
ChIP was performed with 2 μg of the antibody against H3K36me3 (Cat. No. 25250) on sheared chromatin from 1 million HeLaS3 cells. IgG (2 μg/IP) was used as a negative IP control. The immunoprecipitated DNA was analysed by QPCR with optimized PCR primer pairs for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter and for the coding region of the active ACTB gene (figure 1A). The immunoprecipitated 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 1B shows the obtained profiles in genomic regions of chromosome 12 (including the GAPDH positive control), 7 (including the ACTB positive control), 14 and 3, respectively. These results clearly show an enrichment of the H3K36me3 at active genes.

ChIP results obtained with the antibody directed against H3K36me3
ChIP assays were performed using HeLa cells, the antibody against H3K36me3 (Cat. No. 25250) and optimized PCR primer sets for qPCR. ChIP was performed 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. QPCR was performed with primers for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter and for the Sat2 satellite repeat. Figure 2 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K36me3 (Cat. No. 25250) and the crude serum. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the purified antibody was estimated to be 1:19,300.

Cross reactivity tests using the antibody directed against H3K36me3
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K36me3 (Cat. No. 25250) with peptides containing other H3 and H4 modifications and the unmodified sequence. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H3K36me3
Histone extracts (15 μg) from HeLa cells were analysed by Western blot using the antibody directed against H3K36me3 (Cat. No. 25250) 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.

50 µg/56 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human

Affinity purified

synthetic peptide

Aqueous buffer solution

Store at –80°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze/thaw cycles. Aliquots may be stored at –20°C for at least one month.

ChIP/ChIP – seq (1 – 2 µg/ChIP)
ELISA (1:500)
DB (1:20,000 )
WB (1:1000)

The optimal antibody amount per IP should be determined by the end–user. We recommend testing 1–5 µg per IP

Avoid freeze/thaw cycles

Antibodies

Antibody