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Epigenetics



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Human osteosarcoma (U2OS) cells were stained with the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) and with DAPI. Cells were fixed with 2.5% formaldehyde for 30\u2019 and blocked with PBS\/TX-100 containing 1% BSA. Figure A: cells were immunofluorescently labeled with the H3K79me3 antibody (left) diluted 1:300 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right). Figure B and C: staining of the cells with the H3K79me3 antibody after incubation of the antibody with 2 ng\/\u03bcl H4K20me3 and H3K79me3 peptide, respectively.
Histone extracts of HeLa cells (15 \u03bcg) were analyzed by Western blot using the Bioss antibody against H3K79me3 (Cat. No. bs-53121R), diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The molecular weight marker is shown on the right; the location of the protein of interest is indicated on the left.
A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) 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.The figure shows a high specificity of the antibody for the modification of interest.
To determine the titer of the antibody, an ELISA was performed using a serial dilution of Bioss antibody directed against H3K79me3 (Cat. No. bs-53121R), crude serum and flow through in antigen-coated wells. 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 1:3,500.

Catalog #1: bs-53121R

Applications: WB, ELISA, IF, dot-blot

Reactivity: Human, Mouse, Yeast

Conjugate: Unconjugated

Western blot was performed on whole cell (25 \u03bcg, lane 1) and histone extracts (15 \u03bcg, lane 2) from HeLa cells, and on 1 \u03bcg of recombinant histone H3 (lane 3) using H3K79me1 Polyclonal Antibody (Cat. No. bs-53126R). The antibody was diluted 1:200 in TBS-Tween containing 5% skimmed milk.
A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K79me1 (Cat. No. bs-53126R) 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.
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K79me1 (Cat. No. bs-53126R). 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.
ChIP assays were performed using human HeLa cells, the Bioss antibody against H3K79me1 (Cat. No. bs-53126R) 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 \u03bcg of antibody per ChIP experiment was analyzed. IgG (2 \u03bcg\/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. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Catalog #1: bs-53126R

Applications: WB, ELISA, dot-blot, ChIP-seq

Reactivity: Human, Yeast

Conjugate: Unconjugated