H3K9me3 Polyclonal Antibody

HeLa cells were stained with the Bioss antibody against H3K9me3 (bs-53114R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K9me3 antibody (middle) diluted 1:1,000 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The left panel shows staining of the nuclei with DAPI. A merge of both stainings is shown on the right.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against human H3K9me3 (bs-53114R) 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 1:30,000.

A Dot Blot analysis was performed to test the cross-reactivity of the antibody against H3K9me3 (Cat. No. bs-53114R) with peptides containing other modifications and unmodified sequences of histone H3 and H4. 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 1:20,000. The figure shows a high specificity of the antibody for the modification of interest.

ChIP assays were performed using human HeLa cells, the Bioss antibody against H3K9me3 (Cat. No. bs-53114R) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1 million HeLa S3 cells using a ChIP-seq kit. A titration of the antibody consisting of 0.5, 1, 2, and 5 \u00b5g per ChIP experiment was analyzed. IgG (1 \u00b5g\/IP) was used as negative IP control. QPCR was performed with primers for the heterochromatin marker Sat2 and for the ZNF510 gene, used as positive controls, and for the promoters of the active EIF4A2 and GAPDH genes, 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).

ChIP was performed with 0.5 \u00b5g of the Bioss antibody against H3K9me3 (cat. No. bs-53114R) on sheared chromatin from 1,000,000 HeLa cells using a ChIP-seq kit. The IP'd DNA was subsequently analyzed on an Illumina HiSeq 2000. Library preparation, cluster generation, and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure A shows the signal distribution along the long arm of chromosome 19 and a zoom-in to an enriched region containing several ZNF repeat genes. The arrows indicate two satellite repeat regions which exhibit a stronger signal. Figures B, C and D show the enrichment along the ZNF510 positive control target and at the H19 and KCNQ1 imprinted genes.

Lane 1: Human SH-SY5Y cell lysates; Lane 2: Human MOLT4 cell lysates; Lane 3: Human 293T cell lysates probed with Histone H3 (Tri Methyl K9) Polyclonal Antibody, Unconjugated (bs-53114R) at 1:2000 dilution and 4\u00b0C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.