FACE™ EGFR

Figure 1: Detection of EGFR (Y992) phosphorylation using FACE.
The FACE EGFR Kit was used to assay the level of phosphorylated EGFR (Y992) contained within EGF stimulated HeLa cells. FACE assays were performed before and 2, 10 and 30 minutes after EGF induction.

Antibody Specificities

The FACE phospho-EGFR (Tyr992), phospho-EGFR (Tyr1173) and phospho-EGFR (Tyr845) antibodies were raised in rabbits against synthetic phospho-peptides corresponding to residues surrounding phosphorylated Tyr992, Tyr1173 or Tyr845 of human EGF receptor. The phospho-EGFR (Tyr992), phospho-EGFR (Tyr1173) and phospho-EGFR (Tyr845) antibodies recognize EGFR only when phosphorylated at Tyr992, Tyr1173 and Tyr845 respectively. The total-EGFR antibody supplied in the FACE EGFR Kits recognizes EGFR protein regardless of its phosphorylation site.

EGFR Overview

Epidermal Growth Factor (EGF) Receptor (EGFR, HER-1, c-ErbB-1) is a member of the Epidermal Growth Factor Receptor family of Receptor Tyrosine Kinases. These cell surface receptors play an important role in the flow of information from the outside of a cell to the inside. Upon binding of EGF to the extracellular domain, the receptor undergoes dimerization and becomes phosphorylated on several tyrosine residues within the cytoplasmic domain. These result in EGFR activation and increased tyrosine kinase activity toward a variety of intracellular substrates. Autophosphorylation of tyrosine 845, 992 and 1173 are critical to EGFR signaling. Phosphorylation at tyrosine 992 creates a direct binding site for phospholipase C-γ (PLC-γ) resulting in activation of protein kinase C and subsequent downstream signaling cascades. Phosphorylation at 1173 creates a major binding site for the protein tyrosine phosphatase SHP-1, which can dephosphorylate EGFR and thereby block EGFR-induced activation of the ERK1/2 signaling pathway. Tyrosine 845 phosphorylation is mediated by integrin engagement and Src, and regulates receptor function and tumor progression.

FACE™ の使用法

96ウェルプレートで細胞を培養し, 目的の経路を誘導する刺激を与えます。刺激を与えた後, 細胞を固定することでリン酸化を含むタンパク質修飾が保持されます。ブロッキング反応の後, それぞれのウェルに目的タンパク質に特異的な一次抗体, 続いてHRP標識二次抗体を加えます。その後, 発色試薬を加えることにより, 比色定量または化学発光による定量を簡便に行うことができます (図1)。また, キット付属のCrystal Violetを用いて細胞数を補正することができます。それぞれのFACE Kitには目的タンパク質全体とリン酸化型を認識する一次抗体が含まれます。これにより, 細胞数と細胞中の目的タンパク質の総量に対してのリン酸化型タンパク質量を解析することができます。FACEはリン酸化型タンパク質を解析することのできるキットで, 定量的かつ再現性のある結果が得られます。

図1: FACE の操作法

Contents & Storage

Two (or ten) 96-well plates for culturing cells, 96 (or 5 x 96) rxns each of two primary antibodies (1 phospho-specific, 1 specific for native protein), HRP-conjugated secondary antibody, Quenching Solution, 1X Antibody Blocking Buffer, 1X Antibody Dilution Buffer, 10X PBS, 10% Triton X-100, 1% SDS Solution, Developing and Stop Solutions, and Crystal Violet Cell Quantification Solution. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.