H4R3me2(sym) polyclonal antibody

Figure 1. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4R3me2(sym) (cat. No. 15410308) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 1), the titer of the antibody was estimated to be 1:11,750.

Figure 2. Cross reactivity tests using the Diagenode antibody directed against H4R3me2(sym)
To test the cross reactivity of the Diagenode antibody against H4R3me2(sym) (cat. No. 15410308), a Dot Blot analysis was performed with peptides containing other histone arginine methylations and the unmodified H4R3. 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 2 shows the antibody is specific for the H4R3 symmetric dimethylation and also recognizes the H2AR3 symmetric dimethylation.

Figure 3. Western blot analysis using the Diagenode antibody directed against H4R3me2(sym)
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 Diagenode antibody against H4R3me2(sym) (cat. No. C15410308). 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. Immunofluorescence using the Diagenode antibody directed against H4R3me2(sym)
HeLa cells were stained with the Diagenode antibody against H4R3me2(sym) (cat. C15410308) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4R3me2(sym) 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.