Polyclonal antibody raised in rabbit against histone H3 containing the asymmetrically dimethylated arginine 17 (H3R17me2(asym)), using a KLH–conjugated synthetic peptide.

ChIP results obtained with the antibody directed against H3R17me2(asym)
ChIP assays were performed using human osteosarcoma (U2OS) cells, the antibody against H3R17me2(asym) (Cat. # 25275) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1.6 million cells per ChIP reaction. A titration of the antibody consisting of 2, 5, 10 and 15 µl per ChIP experiment was analyzed. IgG (5 µg/IP) was used as negative IP control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 1A: QPCR performed with primers for the GAPDH promoter and for exon 2 of the myoglobin gene.
Figure 1B: QPCR performed with primers for the promoter of the active ALDOA gene and for the coding region of the inactive MYOD gene.

Determination of the titer
To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against H3R17me2(asym) (Cat. # 25275). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:40,000. 

Cross reactivity test using the antibody directed against H3R17me2(asym)
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3R17me2(asym) (Cat. # 25275) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H3R17me2(asym)
Histone extracts of HeLa cells (15 µg) were analyzed by Western blot using the antibody against H3R17me2(asym) (Cat. # 25275) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. 

100 µl

Whole antiserum from rabbit containing 0.05% azide

Human

Whole serum

synthetic peptide

aqueous 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 (10 – 15 µl/ChIP)
ELISA (1:1000 – 1:3000)
DB (1:20,000)
WB (1:250)

The optimal antibody amount per IP should be determined by the end–user. We recommend testing 5–15 µg per IP

Avoid freeze/thaw cycles

Antibodies

Antibody