Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (AB191080)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (AB191080)

ab191080 staining KMT6 / EZH2in 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)

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (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).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (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 (ab191080)

Predicted band size: 85 kDa,92 kDa

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• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (AB191080)

Lane 1 : HEK293 cell lysate (20 μg)

Lane 2 : Mouse spleen tissue lysate (20 μg)

Lane 3 : Rat spleen tissue lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab191080 observed at 93 kDa. Red - loading control, ab8245, observed at 37 kDa.

Human, mouse and rat extracts 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 (ab191080)

Predicted band size: 85 kDa

Observed band size: 93 kDa

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• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (AB191080)

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 (ab191080) 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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• WB

CiteAb

Western blot - Anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal (AB191080)

KMT6 / EZH2 western blot using anti-KMT6 / EZH2 antibody [EPR9307(2)] - N-terminal ab191080. Publication image and figure legend from Xu, L., Feng, J., et al., 2020, Cell Death Dis, PubMed 32341332.

ab191080 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab191080 please see the product overview.

Effects of chidamide on expressions of histone-modifying enzymes (hMOF, SIRT1, and EZH2), and amounts of histone modifications (H4K16ac and H3K27me3) in MM cells.RPMI8226 and H929 cells were treated for 48 h with chidamide at the indicated concentrations. a, d The protein levels of SIRT1 (a), hMOF (b), or EZH2 (d) and amounts of H4K16ac (a) or H3K27me3 (d) were determined by immunoblot with the indicated antibodies. Both H3 and GAPDH were used as controls for protein loading. Blots shown are representative of three independent experiments. b, c, e Relative mRNA levels of SIRT1 (b), hMOF (c), and EZH2 (e) were detected by using quantitative RT-PCR. Mean ± SD of three independent experiments. *p < 0.05, compared with the treatment-naive control.

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