All lanes : Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : EHMT2/G9A knockout HeLa cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 132 kDa

Lanes 1-4: Merged signal (red and green). Green - ab185050 observed at 160 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab185050 Anti-EHMT2/G9A antibody [EPR18894] was shown to specifically react with EHMT2/G9A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265149 (knockout cell lysate ab257080) was used. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab185050 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : EHMT2/G9A knockout HAP1 cell lysate
Lane 3 : HEK293 cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 132 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab185050 observed at 160 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab185050 was shown to recognize EHMT2/G9A when EHMT2/G9A knockout samples were used, along with additional cross-reactive bands. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab185050 and ab8245 (loading control to GAPDH) were diluted 1/1000 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.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on rat kidney is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling EHMT2/G9A with ab185050 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab185050 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

All lanes : Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution

Lane 1 : HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate
Lane 4 : NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 132 kDa
Observed band size: 160,170 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).

Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution + Human fetal heart lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 132 kDa
Observed band size: 160,170 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).

Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 132 kDa
Observed band size: 160,170 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).

All lanes : Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution

Lane 1 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
Lane 2 : Neuro-2a (Mouse neuroblastoma cells) whole cell lysate
Lane 3 : LLC1 (Mouse lung carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 132 kDa
Observed band size: 160,170 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).

All lanes : Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 132 kDa
Observed band size: 160,170 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on epithelial cells of the normal Human colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on tumor cells of the gastric adenocarcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on hepatocytes of the mouse liver is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling EHMT2/G9A with ab185050 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing no staining on MCF7 cell line, as MCF7 cells have a very low level expression of EHMT2/G9A. 

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab185050 at 1/70 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling EHMT2/G9A with ab185050 at 1/150 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

EHMT2/G9A was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab185050 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab185050 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate 10ug (Input).

Lane 2: ab185050 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185050 in HeLa whole cell lysate.

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

Exposure time: 30 seconds.