Anti-EGFR抗体[EP38Y] (ab52894)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 mins before being transferred onto a Nitrocellulose membrane at 30V for 70 mins. 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-labeled 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.

Paraformadehyde-fixed, 0.05% Triton-X permeabilized mouse adult oral epithelia (frozen sections) tissue labeling EGFR (red) using ab52894 at 1/1000 dilution in immunohistochemical analysis.

A blocking step was performed using 5% Gel Block (5% normal donkey serum, 3% BSA, 8% gelatin and 0.1% Triton X-100 in 1X PBS) at 20°C. Primary antibody was incubated for 24 hours at 4°C. Secondary antibody was polyclonal Donkey anti-rabbit Rhodamine Red-X antibody at 1/500 dilution.

Tissues were micro-dissected into ice-cold 1x PBS and fixed for 30 minutes at room temperature (RT) in 4% paraformaldehyde. After washing with PBS 3 times for 10 minutes at RT, samples were equilibrated overnight at 4°C in 15% sucrose solution and then mounted in Tissue-Tek optimal cutting temperature (OCT) compound (Electron Microscopy Services). 12 μm sagittal and coronal sections were cut on a Leica CM1950 cryostat onto Fisher SuperFrost Plus slides and stored at -80°C. Samples were dried at 37°C for 30 minutes before a 1 hour incubation with gelatin block (5% normal donkey serum, 3% BSA, 8% gelatin, and 0.1 Triton X-100 in 1X PBS). Slides were incubated with primary antibodies (Rt-Ki67 and Rb-EGFR) diluted in gelatin block overnight at 4°C and washed 3 times for 5 minutes in 1X PBS at RT. Secondary antibodies (Donkey anti-rat Alexa Fluor^®488 and Donkey anti-rabbit Rhodamine Red-X) were also diluted in gelatin block and added to the slide for 2 hours at RT. DAPI (1/2000) was added to the slide for 5 minutes at RT. Samples were mounted in 100 μl ProLong Gold and covered by glass coverslips.

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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 EDTA buffer, pH 9.0. 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.

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab52894 at 1:250 dilution (0.4 μg/ml).

Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-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.

Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab52894.

Cells were fixed with 4% paraformaldehyde (10 mins) and permeabilized with 90% methanol for 30 mins. 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 mins. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemical anlaysis of rat skin tissue (frozen sections) labeling EGFR using ab52894 at 1/500 dilution.

Tissue was fixed using acetone. Primary antibody was incubated for 16 hours at 8°C using antibody diluent ab64211. Secondary antibody was a Goat anti-Rabbit IgG H&L (Alexa Fluor^®488)(ab150077).

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

Lane 1 (input): HeLa whole cell lysate 10 µg

Lane 2 (+): ab52894 in HeLa whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52894 in HeLa 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

Unpurified ab52894 showing positive staining in endometrial carcinoma tissue.

Unpurified ab52894 stained A431 (Human epidermoid carcinoma cell line) cells.

The cells were fixed in 100% methanol for 5 mins at -20°C and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52894 at 1 in 500) 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 1 hour at room temperature. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with unpurified ab52894 (red line).

The cells were fixed with 80% methanol (5 mins) and then permeabilized with 0.1% PBS-Tween for 20 mins. 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 mins at 22°C. The secondary antibody used was a goat anti-rabbit DyLight^® 488 (IgG H+L) (ab96899) at 1/500 dilution for 30 mins at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1 μg/1x10⁶ cells) used under the same conditions. Unlabeled 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.

Blocking and diluting buffer: 5% NFDM/TBST

Unpurified ab52894 showing positive staining in glioma tissue.

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

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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 cell tissue.

Unpurified ab52894 showing positive staining in renal cell carcinoma tissue.

Unpurified ab52894 showing positive staining in cervical carcinoma tissue.