Anti-EGFR抗体[EP38Y] (ab52894)

Unpurified ab52894 stained A431 cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52894 at 1in500) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Blocking and diluting buffer: 5% NFDM/TBST

ab52894 (purified) at 1:20 dilution (0.5ug) immunoprecipitating EGFR in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 (+): ab52894 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52894 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Blocking and diluting buffer: 5% NFDM/TBST

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with unpurified ab52894 overnight at 4°C in the presence of loading control ab18058 (Mouse monoclonal [SPM227] to Vinculin diluted 1:10000). Antibody binding was detected using IR-labelled goat anti-Rabbit Ab at a 1:10,000 dilution for one hour at room temperature before imaging.

This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein.  Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.

Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labelling EGFR with purified ab52894. Cells were fixed with 4% Paraformaldehyde (10min) and permeabilised with 90% methanol for 30min. Then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by ab52894 at 1/20 dilution (red) for 30 min. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with Purified ab52894 at 1:250 dilution (0.4μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cervical carcinoma tissue sections labeling EGFR with purified ab52894 at 1:100 dilution (0.95 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Unpurified ab52894 showing positive staining in Endometrial carcinoma tissue.

Overlay histogram showing A431 cells stained with unpurified ab52894 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52894, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Unpurified ab52894 showing positive staining in Cervical carcinoma tissue.

Blocking was with 5% BSA incubated for 1 hour at 25°C.

See Abreview

Unpurified ab52894 showing positive staining in Glioma tissue.

Gel running conditions: 4-15%, reduced and denatured.

Blocking agent: 5% milk.

Blocking time: 1 hour.

Incubation (with primary antibody): 16 hours, 4 Celsius.

Dilution buffer: 5%BSA

Unpurified ab52894 showing positive staining in Normal tonsil squamous cells tissue.

Unpurified ab52894 showing positive staining in Renal cell carcinoma tissue.