All lanes : Anti-Dnmt3a antibody [EPR18455-94] (ab227823) at 1/1000 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 - ab227823 observed at 125 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab227823 Anti-Dnmt3a antibody [EPR18455-94] 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. ab227823 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-Dnmt3a antibody [EPR18455-94] (ab227823) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Dnmt3a knockout HAP1 whole cell lysate
Lane 3 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 5 : NIH/3T3 (mouse embryo fibroblast cell line) 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: 102 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

ab227823 was shown to specifically react with wild-type HAP1 cells as signal was lost in Dnmt3a knockout cells. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab227823 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

The observed bands ranging from 75~125kDa represent different Dnmt3a isoforms.

Anti-Dnmt3a antibody [EPR18455-94] (ab227823) at 1/1000 dilution + Rat brain lysate at 20 µg

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

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


Exposure time: 26 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

The observed bands ranging from 75~125kDa represent different Dnmt3a isoforms.

 

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the rat colon (PMID: 24837369) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the mouse colon (PMID: 24837369) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human placenta (PMID: 22413869) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human breast carcinoma (PMID: 24464625) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.