HeLa cells were stained with the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K4me3 antibody (left) diluted 1:100 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.


Western blot was performed on whole cell (40 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3.1, H3.3 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the Bioss antibody against H3K4me3 (Cat. No. bs-53103R). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.


A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. The figure shows a high specificity of the antibody for the modification of interest.


ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 µg of the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) as described above. The IP'd DNA was subsequently analyzed on an Illumina Genome Analyzer. 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. The figure shows the peak distribution along the complete sequence and a 1.2 Mb region of the X-chromosome (figure A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.