Anti-KMT6 / EZH2 antibody [EPR9307(2)] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - BSA and Azide free (AB231165)

ab191080 staining KMT6 / EZH2 in the human cell line Jurkat (human acute T cell leukemia) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/90. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191080).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - BSA and Azide free (AB231165)

Immunofluorescent analysis of HeLa cells (4% Paraformaldehyde-fixed; 0.1% tritonX-100-permeabilized) labeling KMT6 / EZH2 with ab191080 at 1/250 dilution followed by Goat anti rabbit IgG (AlexaFluor® 488) secondary at 1/200 dilution and counter-stained with DAPI (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191080).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - BSA and Azide free (AB231165)

This IHC data was generated using the same anti-KMT6/EZH2 antibody clone, EPR9307(2), in a different buffer formulation (cat# ab191080).

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling KMT6 / EZH2 with ab191080 at 1/250 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin (inset : negative control).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - BSA and Azide free (AB231165)

This WB data was generated using the same anti-KMT6/EZH2 antibody clone, EPR9307(2), in a different buffer formulation (cat# ab191080).

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : KMT6 / EZH2 knockout HAP1 cell lysate (20 μg)
Lane 3 : HEK293 cell lysate (20 μg)
Lane 4 : Ms testis tissue lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab191080 observed at 93 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab191080 was shown to recognize KMT6 / EZH2 when KMT6 / EZH2 knockout samples were used, along with additional cross-reactive bands. Wild-type and KMT6 / EZH2 knockout samples were subjected to SDS-PAGE. ab191080 and ab8245 (loading control to GAPDH) were diluted at 1/500 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 h at room temperature before imaging.

All lanes:

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-epr93072-n-terminal-ab191080'>ab191080</a>)

Predicted band size: 85 kDa,92 kDa

false

• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - BSA and Azide free (AB231165)

False colour image of Western blot : Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab191080 was shown to bind specifically to KMT6 / EZH2. A band was observed at 90 kDa (green arrow) 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 (yellow arrow) 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 [EPR9307(2)] - N-terminal (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-epr93072-n-terminal-ab191080'>ab191080</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

false