All lanes : Anti-Dnmt3L antibody [EPR18774] (ab194094) at 1/1000 dilution

Lane 1 : mESC (Mouse embryonic stem cells ) whole cell lysate
Lane 2 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
Lane 3 : Rat testis lysate
Lane 4 : NTERA-2 cl.D1 [NT2/D1] (Human malignant pluripotent embryonic carcinoma) whole cell lysate
Lane 5 : NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate
Lane 6 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 7 : T-47D (Human ductal breast epithelial tumor 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: 48 kDa
Observed band size: 44 kDa why is the actual band size different from the predicted?

This data was developed using ab194094, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

Observed MW: 48 kDa (mouse), 44 kDa (human).

Exposure times: Lanes 1-2: 1 second; Lanes 3-7: 3 minutes. GAPDH control lanes: 30 seconds.

1. The predicted molecular weight of human Dnmt3L is 44kDa and the predicted molecular weight of mouse Dnmt3L is 48kDa. This is consistent with what has been observed.

2. HeLa and T-47D cells do not express Dnmt3L (PMID: 20460473).

This data was developed using ab194094, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human seminoma tissue labeling Dnmt3L with ab194094 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Mainly nuclear with weak cytoplasmic staining was found on tumor cells of Human seminoma. 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.

This data was developed using ab194094, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Human yolk sac tumor tissue labeling Dnmt3L with ab194094 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining was found on the tumor cells of Human yolk sac tumor. 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.

This data was developed using ab194094, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Dnmt3L with ab194094 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on the Human liver is observed. Counter stained with Hematoxylin. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using ab194094, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Dnmt3L with ab194094 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on the Human liver is observed. Counter stained with Hematoxylin. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using ab194094, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCCIT (Human pluripotent embryonic carcinoma) and HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Dnmt3L with ab194094 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).Confocal image showing nuclear staining on NCCIT cell line. Negative expression in HeLa cells (PMID: 20460473).The nuclear counterstain is DAPI (blue).Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).The negative controls are as follows:--ve control 1: ab194094 at 1/250 dilution followed by ab150120 at 1/1000 dilution.-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

This data was developed using ab194094, the same antibody clone in a different buffer formulation.

Dnmt3L was immunoprecipitated from 1mg of F9 (Mouse embryonic testicular cancer cell line) whole cell lysate with ab194094 at 1/50 dilution.

 Western blot was performed from the immunoprecipitate using ab194094 at 1/1000 dilution.

 VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

 Lane 1: F9 whole cell lysate,10ug (Input).

 Lane 2: ab194094 IP in F9 whole cell lysate.

 Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab191594 in F9 whole cell lysate.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 1 second.