Immunofluorescent analysis of EGFR (pY1068) was performed on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0. 25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ABfinity™ EGFR (pY1068) recombinant rabbit monoclonal antibody (Product # 701053) at a dilution of 1:500 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (Product # A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization of phosphorylated EGFR (pY1068). Panel e shows competition with the phospho-AKT (pT308) peptide. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Human|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 1061–1072 of human EGFR|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:100-1:500|
|Western Blot (WB)||1:500-1:5000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Epidermal Growth Factor Receptor (EGFR) is a 175 kDa transmembrane glycoprotein belonging to tyrosine kinase superfamily. It acts as a receptor for epidermal growth factor (EGF) family proteins. Binding of EGFR to its ligands causes autophosphorylation of tyrosine kinase followed by activation of signal transduction pathways connected to cell proliferation and differentiation. Tyrosine 1068 present within the cytoplasmic domain of the receptor is a major autophosphorylation site that allows binding of Grb2 and activation of the Ras-Raf-ERK1 and ERK2 signaling pathway.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Evaluation of dimerization-inhibitory activities of cyclic peptides containing a ß-hairpin loop sequence of the EGF receptor.
701053 was used in western blot to study the ability of cyclic peptides containing the EGFR beta-hairpin loop sequence to inhibit EGFR dimerization
|Mizuguchi T,Ohara N,Iida M,Ninomiya R,Wada S,Kiso Y,Saito K,Akaji K||Bioorganic and medicinal chemistry (20:5730)||2012|