Recombinant 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 (blue), and 20µl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (yellow).  The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

ChIP was performed according to the literature (PMID: 21880597).

Merged signal (red and green). Green - ab191250 observed at 93 kDa. Red - 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 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 (ab216773) and 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.

Blocking/Dilution buffer: 5% NFDM/TBST.

Blocking/Dilution buffer: 5% NFDM/TBST.

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) (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 (ab172730) instead of ab191250 in HeLa whole cell lysate.

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

Exposure time: 3 minutes.