Intracellular Staining by Flow Cytometry
|Detection of Phospho-EGFR in EGF-treated A431 Human Cell Line by Flow Cytometry. A431 human epithelial carcinoma cell line was untreated (open histogram), or treated for 5 minutes with 100 ng/mL Recombinant Human EGF (Catalog # 236-EG; filled histogram) then stained with Rabbit Anti-Human Phospho-EGFR (Y845) Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF3394), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). Rabbit IgG (Catalog # AB‑105‑C, data not shown) was used as a control antibody. To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
|Detection of Human Phospho-EGFR (Y845) by Western Blot. Western blot shows lysates of A431 human epithelial carcinoma cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human EGF (Catalog # 236-EG) for 5 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human Phospho-EGFR (Y845) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3394), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-EGFR (Y845) at approximately 175 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
|Phospho-EGFR (Y845) in A431 Human Cell Line. EGFR phosphorylated at Y845 was detected in immersion fixed A431 human epithelial carcinoma cell line untreated (lower panel) or treated (upper panel) with pervanadate using Rabbit Anti-Human Phospho-EGFR (Y845) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3394) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
Epidermal growth factor receptor (EGFR, also known as ErbB1/HER1) is the founding member of the EGFR family of receptor tyrosine kinases. Ligand binding induces receptor dimerization and autophosphorylation on multiple tyrosine residues. Phosphorylation of tyrosine 845 is associated with regulation of receptor function and tumor progression.
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