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Synthetic phosphopeptide (Human) derived from the region of EGFR that contains tyrosine 1068.
Our Abpromise guarantee covers the use of ab5644 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. Detects a band of approximately 185 kDa.|
Ab5644 staining EFGR in 70% confluent log phase A-431 cells treated with 200ng/ml of EGF for 10 minutes by ICC/IF (Immunocytochemistry/Immunofluorescence). The cells were fixed with 4% paraformaldehyde for 10 minutes; permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 1% BSA for hour at room temperature. Primary antibody was used at 1/100 dilution. A Goat anti-rabbit IgG (H+L) Superclonal, Alexa Fluor® 488 conjugate was used as the secondary antibody at 1/2000 dilution (image a). Nuclei (image b) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (C) was stained with Rhodamine Phalloidin. Image d represents the merged image showing membrane localization. Image e represents cells treated with antagonist, Afatinib (1µM for 6hrs) followed by EGF (200 ng/ml for 10 minutes), showing no Phospho-EGFR staining. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Western blot analysis using ab5644 shows increased expression of proteins phosphorylated at the tyrosine residues in A-431 and A549 cell lines upon EGF treatment and pre-treatment with EGFR-antagonists, Gefitinib and Afatinib. This results in inhibition of Phospho-EGFR in A-431 and A549 cell lines