Lane 1 : Anti-TGF alpha antibody [EPR15346] (ab208156) at 1/1000 dilution
Lane 2 : Anti-TGF alpha antibody [EPR15346] (ab208156) at 1/5000 dilution

All lanes : MCF-7 whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 17 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

The different result is due to the lysates preparation method. The lysate in the left image is prepared by RIPA lysis method. The lysate in the right image is prepared by 1% SDS Hot lysis method. This antibody works better in 1% SDS Hot Lysates in WB.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here.

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling TGF alpha with ab208156 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on Human liver is observed. 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling TGF alpha with ab208156 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody  -Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG  (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab208156 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.

TGF alpha was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate with ab208156 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab208156 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HepG2 whole cell lysate 10µg (Input).

Lane 2: ab208156 IP in HepG2 whole cell lysate.

Lane 3: Rabbit IgG,monoclonal - Isotype Control (ab172730) instead of ab208156 in HepG2 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

All lanes : Anti-TGF alpha antibody [EPR15346] (ab208156) at 1/1000 dilution

Lane 1 : Human fetal skin lysate
Lane 2 : Human fetal liver lysate
Lane 3 : Human breast cancer lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 17 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 3: 3 minutes; Lane 2: 1 minute.

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labeling TGF alpha with ab208156 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on the cancer cells of Human hepatocellular carcinoma is observed. 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells, treated with Brefeldin A (10 µM, 18 hours), labeling TGF alpha with ab208156 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cells, treated with Brefeldin A (10 µM, 18 hours). The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG  (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab208156 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.

TGF alpha was immunoprecipitated from 1mg of Ramos (Human Burkitt's lymphoma cell line) whole cell lysate with ab208156 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab208156 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Ramos whole cell lysate, 10µg (Input).

Lane 2: ab208156 IP in Ramos whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab208156 in Ramos whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

All lanes : Anti-TGF alpha antibody [EPR15346] (ab208156) at 1/1000 dilution

Lane 1 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 17 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 15 seconds; Lane 2: 3  minutes.