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

ChIP results obtained with the monoclonal antibody directed against H4K20me1
ChIP assays were performed using human HeLa cells, the monclonal antibody against H4K20me1 (Cat. # 25279) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 2, 5, and 10 µg per ChIP experiment was analyzed. IgG (5 µg/IP) was used as negative IP control. QPCR was performed with primers for the coding region of the active GAPDH and the inactive TSH2B gene, for the GAPDH promoter and for a region located 1 kb upstream of the GAPDH promoter. 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 monoclonal antibody directed against H4K20me1
ChIP was performed as described above using 1 µg of the antibody against H4K20me1 (Cat. # 25279). The immunoprecipitated DNA was analyzed 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 in genomic regions from chromosomes 12, 7, 14 and 3 surrounding the GAPDH, ACTB, FOS and EIF4A2 positive controls gene (figure 2A, B, C and D, respectively).

Cross reactivity tests using the monoclonal antibody directed against H4K20me1
To check the specificity of the monoclonal antibody against H4K20me1 (cat. # 25279) 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 monoclonal antibody directed against H4K20me1
Histone extracts of HeLa cells (15 µg) were analyzed by Western blot using the monoclonal antibody against H4K20me1 (cat. # 25279) diluted 1:200 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.

Immunofluorescence using the monoclonal antibody directed against H4K20me1
Human osteosarcoma (U2OS) cells were stained with the antibody against H4K20me1 (Cat. # 25279) and with DAPI. Cells were fixed with ice cold methanol for 10 min. and blocked with PBS/TX-100 containing 5% normal goat serum. Figure 5A: cells were immunofluorescently labeled with the H4K20me1 antibody (left), diluted 1:600 in blocking solution followed by an anti-mouse antibody conjugated to Alexa488 or with DAPI (right), which specifically labels DNA. Figure 5B: staining of the cells with the H4K20me1 antibody after incubation of the antibody with 5 ng/µl of the corresponding blocking peptide.

50 µg/23 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human

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

Protein A 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)
DB (1:20,000)
IF (1:600)
WB (1:200)

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