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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human EGFR. Synthetic phospho-peptide corresponding to residues surrounding Tyr1068 of mature human EGFR.
Database link: P00533
A trial size is available to purchase for this antibody.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Our Abpromise guarantee covers the use of ab52894 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/1000 - 1/10000. Detects a band of approximately 175 kDa (predicted molecular weight: 134 kDa).Can be blocked with EGFR peptide (ab204282).
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
For unpurified use at 1/50.
For unpurified use at 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples.
|ICC/IF||1/250 - 1/500.|
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 labeling EGFR with purified ab52894 at 1:20 dilution (5 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - 90% methanol. Unlabeled control - Rabbit monoclonal IgG (Black).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.
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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"