Anti-KMT6 / EZH2 antibody [EPR25353-284]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labeling KMT6 / EZH2 with ab307646 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Immunohistochemical analysis of paraffin-embedded human pancreatic adenocarcinoma and adjacent tissue labeling KMT6 / EZH2 with ab307646 at 1/100 dilution (5.43 µg/ml) followed by 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 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling KMT6 / EZH2 with ab307646 at 1/100 dilution (5.43 µg/ml) followed by 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 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Immunohistochemical analysis of paraffin-embedded cell pellets labeling KMT6 / EZH2 with ab307646 at 1/1000 dilution (0.543 µg/ml) followed by 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 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) (Right panel) / KMT6 knockout HAP1 (Left panel) cells labeling KMT6 / EZH2 with ab307646 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled 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.

• IP

Supplier Data

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

KMT6 / EZH2 was immunoprecipitated from 0.35 mg NCCIT (human pluripotent embryonic carcinoma epithelial cell) whole cell lysate 10 µg with ab307646 at 1/30 dilution (2µg 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 whole cell lysate 10 µg 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.

All lanes:

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR25353-284] (ab307646) at 1/30 dilution

All lanes:

NCCIT (human pluripotent embryonic carcinoma epithelial cell)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 85 kDa

false

Exposure time: 5s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling KMT6 / EZH2 with ab307646 at 1/1000 dilution (0.543 µg/ml) followed by 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 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling KMT6 / EZH2 with ab307646 at 1/1000 dilution (0.543 µg/ml) followed by 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 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized B16-F10 (Mouse skin melanoma) cells labeling KMT6 / EZH2 with ab307646 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

Immunohistochemical analysis of paraffin-embedded mouse pancreatic tumor tissue labeling KMT6 / EZH2 with ab307646 at 1/1000 dilution (0.543 µg/ml) followed by 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 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.

• IP

Supplier Data

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

KMT6 / EZH2 was immunoprecipitated from 0.35 mg B16-F10 (mouse skin melanoma cell) whole cell lysate 10 µg with ab307646 at 1/30 dilution (2µg 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 whole cell lysate 10 µg 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.

All lanes:

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [EPR25353-284] (ab307646) at 1/30 dilution

All lanes:

B16-F10 (mouse skin melanoma cell)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 85 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

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

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

All lanes:

Western blot - Anti-KMT6 / EZH2 antibody [EPR25353-284] (ab307646) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervical adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 85 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - 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.

All lanes:

Western blot - Anti-KMT6 / EZH2 antibody [EPR25353-284] (ab307646) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg

Lane 2:

KMT6 / EZH2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

B16-F10 (mouse skin melanoma cell) whole cell lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Observed band size: 85 kDa

true

• WB

Supplier Data

Western blot - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

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.

All lanes:

Western blot - Anti-KMT6 / EZH2 antibody [EPR25353-284] (ab307646) at 1/1000 dilution

All lanes:

NCCIT (human pluripotent embryonic carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 85 kDa

false

Exposure time: 3s

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells and 5 µg of ab307646 [EPR25353-284]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells and 5 µg of ab307646 [EPR25353-284]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-KMT6 / EZH2 antibody [EPR25353-284] (AB307646)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells and 5 µg of ab307646 [EPR25353-284]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.