H3K79me2 Polyclonal Antibody

A Dot Blot analysis was performed to test the cross reactivity of the H3K79me2 antibody (bs-53039R) with peptides containing other modifications of histone H3. Other H3 modifications include mono- and trimethylation of the same lysine and mono-, di- and trimethylation of lysine 9, 27 and 36. 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 1:50,000. The figure shows a high specificity of the antibody for the modification of interest.

ChIP assays were performed using human osteosarcoma (U2OS) cells, the H3K79me2 antibody (bs-53039R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 2, 7 and 15 \u03bcl per ChIP experiment was analysed. Additionally, ChIP was performed with 2 and 15 \u03bcl of antibody after incubation with 5 nmol blocking peptide for 1 hour at room temperature. IgG (5 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the ALDOA (fructose-bisphosphate aldolase A) promoter and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

To determine the titer, an ELISA was performed using a serial dilution of the H3K79me2 antibody (bs-53039R). 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 1:4,500.