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Applications: ChIP (2 µg/IP)
ELISA (1:500)
Dot (1:20,000)
WB (1:200)
Assay Conditions: ChIP results obtained with the antibody directed against H3K79me1
ChIP assays were performed using human HeLa cells, the antibody against H3K79me1 (Cat. # 25261) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million 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 specific for a region 1 kb upstream of the promoter and the coding region of the active GAPDH gene, used as positive controls, and for exon 2 of the inactive myoglobin (MB) 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 H3K79me1
ChIP was performed with 1 µg of the antibody against H3K79me1 (Cat. # 25261) as described above and the immunoprecipitated DNA was subsequently 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 along the complete sequence and a 1 Mb region of the X-chromosome (figure 2A and B), in 100 kb regions surrounding the GAPDH positive control and EIF4A2 genes (figure 2C and D).Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K79me1 (Cat. # 25261). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:11,500.Cross reactivity test using the antibody directed against H3K79me1
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K79me1 (Cat. # 25261) with peptides containing other histone modifications and the unmodified H3K79. 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 H3K79me1
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 H3 (lane 3) using the antibody against H3K79me1 (Cat. # 25261). The antibody was 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.
Background: Methylation of H3K79 is usually catalyzed by the methyltransferase Dot1L and is associated with gene transcription.
Concentration: 50 µg/31 µl
Description: Polyclonal antibody raised in rabbit against histone H3 containing the monomethylated lysine 79 (H3K79me1), using a KLH-conjugated synthetic peptide.
Format: Aqueous buffer solution
Formulation: PBS containing 0.05% azide and 0.05% ProClin 300
Immunogen: synthetic peptide
Purification: Affinity purified
Storage Stability: 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.
Uniprot: P68431
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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