Recombinant Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491)

Immunohistochemical analysis of paraffin-embedded sections labeling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/200 dilution (2.345 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.
Panel A: HEK-293T cells transfected with a human EZH2 Y646N expression vector containing a myc-His tag.
Panel B: HEK-293T cells transfected with a human EZH2 WT expression vector containing a myc-His tag.
Panel C: HEK-293T transfected with an empty vector.
Positive staining on HEK-293T cells transfected with a human EZH2 Y646N expression vector (Panel A), no staining on HEK-293T cells transfected with a human EZH2 WT expression vector (Panel B), no staining on HEK-293T transfected with an empty vector (Panel C). The section was incubated with ab300491 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/200 dilution (2.345 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.
Negative control: No staining on human testis.
The section was incubated with ab300491 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Blocking / Diluting buffer: 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate.

Loading controls
All Lanes:  ab191250, Recombinant Anti-KMT6 / EZH2 antibody and ab213204, Recombinant Anti-6X His tag^® antibody

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK293T (human embryonic kidney epithelial cells) labelling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/2000 dilution (green). Confocal image showing nuclear staining in HEK-293T cells transfected with a human EZH2 Y646N expression vector containing a myc tag. An anti Myc-Tag Mouse monoclonal antibody (Alexa Fluor^® 647) at 1/100 dilution was used as a counterstain (red). The nuclear counterstain is DAPI (blue).

Flow Cytometry (Intracellular) analysis of HEK-293T (human embryonic kidney) cells transfected with a human wild type KMT6 / EZH2 expression vector containing a myc-his tag (Left) / HEK-293T transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag (Right). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% Methanol. ab300491 used at a 1/500 dilution. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution.

KMT6 / EZH2 (mutated Y646N) was immunoprecipitated from 10 μg HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate with ab300491 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab300491 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate 10 μg (Input).

Lane 2: HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300491 in HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate.

Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.

Observed MW(KDa): 98

This blot was developed using a high sensitivity ECL substrate.

Dot Blot - Anti KMT6 / EZH2 (mutated Y646N)

Concentration of ab300491: 1/1000 dilution (0.469 μg/ml)

Secondary ab: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051), 1/100000 dilution

Blocking/diluting buffer: 5% NFDM/TBST

Lane 1: Human KMT6 / EZH2 mutated Y646N peptide

Lane 2: Human wild type KMT6 / EZH2 peptide

Exposure time: 3 minutes