Recombinant Anti-KMT6 / EZH2 antibody [EPR25353-284] (ab307646)

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

The samples were run on a Bis-Tris gel.

Performed under reducing conditions.      

False colour image of Western blot: Anti-KMT6 / EZH2 antibody (ab307646) 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, ab307646 was shown to bind specifically to KMT6 / EZH2. A band was observed at 85kDa in wild-type HAP1 cell lysates whereas no signal observed at this size in KMT6 / EZH2 knockout cell line. To generate this image, wild-type and KMT6 / EZH2 knockout HAP1 cell lysates were analyzed. First, samples were run on a Bis-Tris gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Exposure time: 26 seconds

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The bands beneath the target band (85 kDa) are likely to be degraded target fragments.

Exposure time: 3 seconds

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling KMT6 / EZH2 with ab307646 at 1/100 (5.43 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human colon. The section was incubated with ab307646 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human pancreatic adenocarcinoma and adjacent tissue labeling KMT6 / EZH2 with ab307646 at 1/100 (5.43 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human pancreatic adenocarcinoma (image A) and no staining on the adjacent tissue (image B). The section was incubated with ab307646 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling KMT6 / EZH2 with ab307646 at 1/1000 (0.543 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse colon. The section was incubated with ab307646 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse pancreatic tumor tissue labeling KMT6 / EZH2 with ab307646 at 1/1000 (0.543 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse pancreatic tumor. The section was incubated with ab307646 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling KMT6 / EZH2 with ab307646 at 1/1000 (0.543 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat colon. The section was incubated with ab307646 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded A) Wild-type HAP1 (Human chronic myelogenous leukemia near-haploid cell) cell pellet and B) EZH2 knockout HAP1 cell pellet labeling KMT6 / EZH2 with ab307646 at 1/1000 (0.543 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on (A) wild-type HAP1 cell pellet, no staining on (B) EZH2 knockout HAP1 cell pellet. The section was incubated with ab307646 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized EZH2 KO HAP1 (EZH2 knockout human chronic myelogenous leukemia) cells labelling KMT6 / EZH2 with ab307646 at 1/50 (10.86 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in Parental HAP1 cell line, and no taining in EZH2 KO HAP1 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labelling KMT6 / EZH2 with ab307646 at 1/50 (10.86 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in NCCIT cell line .Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized B16-F10 (mouse skin melanoma cell) cells labelling KMT6 / EZH2 with ab307646 at 1/50 (10.86 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in B16-F10 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right) / KMT6 knockout HAP1(Left) cells labelling KMT6 / EZH2 with ab307646 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labelling KMT6 / EZH2 with ab307646 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized B16-F10 (Mouse skin melanoma) cells labelling KMT6 / EZH2 with ab307646 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

KMT6 / EZH2 was immunoprecipitated from 0.35 mg NCCIT (human pluripotent embryonic carcinoma epithelial cell) whole cell lysate 10 ug with ab307646 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307646 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NCCIT (human pluripotent embryonic carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab307646 IP in NCCIT whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab307646 in NCCIT whole cell lysate

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

Exposure time: 5 seconds

Lysate was freshly made and used for IP immediately to minimize protein degradation.

KMT6 / EZH2 was immunoprecipitated from 0.35 mg B16-F10 (mouse skin melanoma cell) whole cell lysate 10 ug with ab307646 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307646 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: B16-F10 (mouse skin melanoma cell) whole cell lysate 10 ug

Lane 2: ab307646 IP in B16-F10 whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab307646 in B16-F10 whole cell lysate

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

Exposure time: 5 seconds

Lysate was freshly made and used for IP immediately to minimize protein degradation.

Chromatin was prepared from NCCIT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab307646(red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).