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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK293T (human embryonic kidney epithelial cell) cells labelling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/50 (9.38ug/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 HEK-293T cells transfected with a human EZH2 Y646N expression vector containing a myc tag. is observed. Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 0.38ug/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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/200 (2.345 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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) was used.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

Immunohistochemical analysis of paraffin-embedded Panel A HEK-293T ce tissue labeling KMT6 / EZH2 (mutated Y646N) with ab300491 at 1/200 (2.345 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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) was used.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

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.

• IP

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Immunoprecipitation - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

KMT6 / EZH2 (mutated Y646N) was immunoprecipitated from 0.35 mg HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate 10 ug with ab300491 at 1/30 dilution (2ug in 0.35mg lysates).

Western blot was performed on the immunoprecipitate using ab300491 at 1/1000 dilution.

VeriBlot for IP secondary antibody (HRP) (ab131366) was used 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 ug

Lane 2 : ab300491 IP in 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 and concentration : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491) at 1/1000 dilution

All lanes:

HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containing a myc-his tag, whole cell lysate at 10 µg

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

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Exposure time: 3min

• WB

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Western blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (ab300491) at 1/1000 dilution

Lane 1:

HEK-293T cells transfected with a human KMT6 / EZH2 mutated Y646N expression vector containi a myc-his tag, whole cell lysate, 10

Lane 2:

HEK-293T cells transfected with a human wild type KMT6 / EZH2 expression vector containi a myc-his tag, whole cell lysate, 10

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: 98 kDa

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Exposure time: 6s

• Dot

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Dot Blot - Anti-KMT6 / EZH2 (mutated Y646N) antibody [EPR24904-99] (AB300491)

Dot blot analysis of KMT6 / EZH2 (mutated Y646N) using ab300491 at 1 : 1000 (0.469 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Exposure time : 3 minutes Blocking and diluting buffer and concentration : 5% NFDM/TBST