All lanes : Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : Dnmt3a knockout HeLa cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 102 kDa

Lanes 1-4: Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab188470 Anti-Dnmt3a antibody [EPR18455] was shown to specifically react with Dnmt3a in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261793 (knockout cell lysate ab257128) was used. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab188470 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 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.

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human placenta tissue sections labeling Dnmt3a with purified ab188470 at 1/2000 (0.409 µg/ml). Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/5000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Dnmt3a knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 102 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab188470 was shown to recognize Dnmt3a when Dnmt3a knockout samples were used, along with additional cross-reactive bands. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab188470 and ab8245 (loading control to GAPDH) were diluted to 1/5000 and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Dnmt3a with purified ab188470 at 1/80 dilution(10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

All lanes : Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/5000 dilution

Lane 1 : HeLa whole cell lysate
Lane 2 : Mouse skin tissue lysate
Lane 3 : Rat skin tissue lysate
Lane 4 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 102 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

 

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab188470 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labeling Dnmt3 with purified ab188470 at 1/2000 (0.409 µg/ml). Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue sections labeling Dnmt3a with purified ab188470 at 1/2000 (0.409 µg/ml). Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain.

Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/10000 dilution + HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 102 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (J Biol Chem. 2002. 277, 38746-38754. PMID: 2138111).

Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/10000 dilution + HEK-293 (Human epithelial cells from embryonic kidney) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 102 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (J Biol Chem. 2002. 277, 38746-38754. PMID: 2138111).

All lanes : Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/2000 dilution

Lane 1 : Rat brain lysate
Lane 2 : Rat heart lysate
Lane 3 : C6 (Rat glial tumor cells) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 102 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Dnmt3a with ab188470 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 and weakly cytoplasmic staining on HeLa cell line. The nuclear counter stain 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: ab188470 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.