All lanes : Anti-ErbB2 / HER2 (phospho Y1248) + ErbB4 / HER4 (phospho Y1284) antibody [EPR19547] (ab201013) at 1/1000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes
Lane 3 : Untreated A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 4 : A431 (Human epidermoid carcinoma cell line) whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes

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: 138, 147 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

This data was developed using ab201013, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-2: 5 seconds; Lanes 3-4: 10 seconds.

This target could be induced to increase by EGF as proved by literatures (PMID: 18945363; 17030621).

The MW is also consistent with some literature (PMID: 18180459).

All lanes : Anti-ErbB2 / HER2 (phospho Y1248) + ErbB4 / HER4 (phospho Y1284) antibody [EPR19547] (ab201013) at 1/5000 dilution

Lane 1 : Untreated SK-BR-3 (human mammary gland adenocarcinoma ) whole cell lysate
Lane 2 : SK-BR-3(human mammary gland adenocarcinoma ) whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes

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: 138, 147 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

This data was developed using ab201013, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

This target could be induced by EGF as shown by literature (PMID: 18945363; 17030621).

The MW is also consistant with that shown within literature (PMID: 18180459).

This data was developed using ab201013, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ERBB2 / HER2 (phospho Y1248) + ERBB4 / HER4 (phospho Y1284) with ab201013 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased cytoplasmic staining after EGF treatment (200ng/ml; 30min). 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: ab201013 at 1/100 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 ab201013, the same antibody clone in a different buffer formulation.

>Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ERBB2 / HER2 (phospho Y1248) + ERBB4 (phospho Y1284) with ab201013 at 1/50 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor

This data was developed using ab201013, the same antibody clone in a different buffer formulation.ERBB2 / HER2 (phospho Y1248) + ERBB4 / HER4 (phospho Y1284) were immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate, starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes, with ab201013 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab201013 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution. Lane 1: HeLa whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes 10µg (Input). Lane 2: ab201013 IP in HeLa whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes. Lane 3: Rabbit IgG,monoclonal [EPR25A]- Isotype Control (ab172730) instead of ab201013 in HeLa whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes. Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 1 second.

This data was developed using ab201013, the same antibody clone in a different buffer formulation.ERBB2 / HER2 (phospho Y1248) + ERBB4 / HER4 (phospho Y1284) were immunoprecipitated from 0.35 mg of A431 (Human epidermoid carcinoma cell line) whole cell lysate, starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes, with ab201013 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab201013 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution. Lane 1: A431 whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes 10µg (Input). Lane 2: ab201013 IP in A431 whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab201013 in A431 whole cell lysate starved 4 hours, then treated with 200 ng/ml EGF for 15 minutes. Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 1 second.