Recombinant Anti-ErbB2 / HER2 (phospho Y1248) + ErbB4 / HER4 (phospho Y1284) antibody [EPR19547] (ab201013)

Dot blot analysis using 1/1000 dilution ab201013 and Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051)  secondary at 1/100000 dilution. 

Blocking and diluting buffer: 5% NFDM/TBST

Lane 1: ErbB4 non-phospho peptide

Lane 2: ErbB4 Y1284 phospho peptide

Lane 3: ErbB2 non-phospho peptide

Lane 4: ErbB2 Y1248 phospho peptide

Exposure time: 3 minutes

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?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time:  Lane 1 and 2: 5 seconds; Lane 3 and 4: 10 seconds.

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

The MW is also consistent with some literatures (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

Blocking/Diluting buffer 5% NFDM/TBST. 

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

The MW is also consist with some literatures showed(PMID: 18180459)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ERBB2 (phospho Y1248) + ERBB4 (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.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ERBB2 (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 Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody. 

 

 

ERBB2 (phospho Y1248) + ERBB4 (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.

ERBB2 (phospho Y1248) + ERBB4 (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.