Polyclonal antibody raised in rabbit against the region of histone H3 containing the asymmetrically dimethylated R17 and the acetylated lysine 18 (H3R17me2(asym)K18ac), using a KLH–conjugated synthetic peptide.

ChIP results obtained with the antibody directed against H3R17me2(asym)K18ac
ChIP assays were performed using human HeLa cells, the antibody against H3R17me2(asym)K18ac (cat. No. 25276) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1,000,000 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 for the promoters of the active EIF4A2 and c-fos genes, used as positive controls and for the inactive MYOD1 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).

ChIP-seq results obtained with the antibody directed against H3R17me2(asym)K18ac
ChIP was performed as described above using 1 µg of the antibody against H3R17me2(asym)K18ac (cat. No. 25276). 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 2 shows the peak distribution along the complete human X-chromosome and a zoomin to a 500 kb region (figure 2A and B), and in two regions on chromosome 14 and 3 surrounding the c-fos and EIF4A2 positive control genes (figure 2C and D, respectively).

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against H3R17me2(asym)K18ac (cat. No. 25276). 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 antibody was estimated to be 1:28,800.

Cross reactivity tests using the antibody directed against H3R17me2(asym)K18ac
To test the cross reactivity of the antibody against H3R17me2(asym)K18ac (cat. No. 25276), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3R17K18. One hundred to 0.2 pmol of the respective peptides 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 H3R17me2(asym)K18ac
Western blot was performed on whole cell (25 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 antibody against H3R17me2(asym)K18ac (cat. No. 25276). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.

Immunofluorescence using the antibody directed against H3R17me2(asym)K18ac
Mouse NIH3T3 cells were stained with the antibody against H3R17me2(asym)K18ac (cat. No. 25276) and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3R17me2(asym)K18ac antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. 

50 µg/53 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human, mouse, broad range

Highly specific antibody for H3R17me2(asym)K18ac. Does not cross–react with H3R17me2(asym) or H3K18ac.

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 µg/IP)
ELISA (1:1000)
DB (1:20,000)
WB (1:500)
IF (1:500)

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