Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade

5 product images

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  ChIP - Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab195477)

  ChIP results obtained with ab195477 directed against Histone H3 (tri methyl K27).
  

  ChIP assays were performed using human HeLa cells, ab195477 and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes EIF4A2 and GAPDH as negative controls, and for the coding regions of the inactive genes MYT1 and TSH2B as positive controls. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K27me3 is preferably present at inactive genes.

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  ChIP-sequencing - Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab195477)

  ChIPseq results obtained with ab195477 directed against Histone H3 (tri methyl K27).
  

  ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 μg of ab195477. The IP'd DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure shows the enrichment in genomic regions of chromosome 6, surrounding the TSH2B gene (indicated by an arrow; fig A), of chromosome 20, surrounding the MYT1 gene (fig B), and of chromosome 2 and 3 (figure C and D).

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab195477)

  Immunofluorescent analysis of NIH 3T3 cells labeling Histone H3 (tri methyl K27) with ab195477 at 1/200 dilution. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with ab195477 in blocking solution followed by an ant-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.

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  Dot Blot - Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab195477)

  Dot Blot analysis was performed to test the cross reactivity of ab195477 against Histone H3 (tri methyl K27) with peptides containing other modifications of Histone H3 and H4 and the unmodified H3K27 sequence. 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 1/5000. Figure shows a high specificity of the antibody for the modification of interest. Please note that that antibody also recognizes the modification if S28 is phosphorylated.

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  Western blot - Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab195477)

  Histone H3 (tri methyl K27) Western blot staining using rabbit Anti-Histone H3 (tri methyl K27) antibody

  The antibody was diluted in TBS-Tween containing 5% skimmed milk.

  All lanes: Western blot - Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab195477) at 1/500 dilution

  Lane 1: HeLa whole cell extract at 25 µg

  Lane 2: HeLa histone extract at 15 µg

  Lane 3: Recombinant Histone H2A at 1 µg

  Lane 4: Recombinant Histone H2B at 1 µg

  Lane 5: Recombinant Histone H3 at 1 µg

  Lane 6: Recombinant Histone H4 at 1 µg

  Predicted band size: 15 kDa

  Observed band size: 15 kDa