Polyclonal antibody raised in rabbit against histone H4 containing the monomethylated lysine 20 (H4K20me1), using a KLH–conjugated synthetic peptide

ChIP results obtained with the antibody directed against H4K20me1
ChIP assays were performed using human HeLa cells, the antibody against H4K20me1 (cat. No. 25280) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the coding region of the active GAPDH and ACTB genes, used as positive controls, and for the GAPDH promoter and a region located 1 kb upstream of the GAPDH promoter, 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).

Determination of the antibody titer
To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against H4K20me1 (cat. No. 25280) 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 2), the titer of the antibody was estimated to be 1:51,100.

Cross reactivity tests using the antibody directed against H4K20me1
To check the specificity of the antibody against H4K20me1 (cat. No 25280) a Dot Blot was performed with peptides containing different modifications of histone H3 and H4 or the unmodified H4K20 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 3 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H4K20me1
Western blot was performed on whole cell extracts (25 μg, lane 1) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 2, 3, 4 and 5, respectively) using the antibody against H4K20me1 (cat. No. 25280). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left, the position of the protein is indicated on the right.

50 µg/25 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human

Specific for H4K20me1. Does not cross–react with H4K20me2, H4K20me3, or H4K20un.

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 (1 – 2 µg/IP)ELISA (1:1000)
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