Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade

6 product images

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  ChIP - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  Chromatin was prepared from MDA-MB-231 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 5μg of ab191250 (red), and 20μl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  

  Cell line and primers selection based on literature (PMID: 21880597).

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  Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  Merged signal (red and green). Green - ab191250 observed at 93 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  ab191250 was shown to specifically recognize KMT6/EZH2 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when KMT6/EZH2 knockout samples were examined. Wild-type and KMT6/EZH2 knockout samples were subjected to SDS-PAGE. ab191250 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250) at 1/1000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: KMT6 / EZH2 knockout HAP1 cell lysate at 20 µg

  Lane 3: HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

  Lane 4: MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

  Predicted band size: 85 kDa

  Observed band size: 90 kDa

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  Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  KMT6 / EZH2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab191250 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab191250 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10000 dilution.
  

  Lane 1: HeLa whole cell lysate 10 μg (Input).

  Lane 2: ab191250 IP in HeLa whole cell lysate.

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab191250 in HeLa whole cell lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 3 minutes.

  All lanes: Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  Predicted band size: 85 kDa

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  Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250) at 1/5000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

  Lane 2: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 85 kDa

  Observed band size: 90 kDa

  Exposure time: 30s

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  Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250) at 1/1000 dilution

  All lanes: U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 85 kDa

  Observed band size: 90 kDa

  Exposure time: 30s

• 

  Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250)

  False colour image of Western blot: Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab191250 was shown to bind specifically to KMT6 / EZH2. A band was observed at 90 kDa in wild-type MCF7 cell lysates with no signal observed at this size in ezh2 CRISPR-Cas9 edited cell line ab281611 (CRISPR-Cas9 edited cell lysate ab282963). The band observed in the CRISPR-Cas9 edited lysate lane below 90 kDa is likely to represent a truncated form of KMT6 / EZH2. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ezh2 CRISPR-Cas9 edited MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (ab191250) at 1/1000 dilution

  Lane 1: Wild-type MCF7 cell lysate at 20 µg

  Lane 2: ezh2 CRISPR-Cas9 edited MCF7 cell lysate at 20 µg

  Lane 3: HEK-293 cell lysate at 20 µg

  Lane 4: Mouse Testis cell lysate at 20 µg

  Performed under reducing conditions.

  Observed band size: 90 kDa