Anti-KMT6 / EZH2 antibody [EPR20108] - BSA and Azide free

• IP

Supplier Data

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR20108] - BSA and Azide free (AB240992)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191250).

All lanes:

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-epr20108-chip-grade-ab191250'>ab191250</a>)

Predicted band size: 85 kDa

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• WB

Supplier Data

Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - BSA and Azide free (AB240992)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191250).

All lanes:

Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - ChIP Grade (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-epr20108-chip-grade-ab191250'>ab191250</a>) 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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• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [EPR20108] - BSA and Azide free (AB240992)

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] (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 (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-epr20108-chip-grade-ab191250'>ab191250</a>) 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

Observed band size: 90 kDa

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