FACE™ c-Src

in-cell Western analysis for phospho c-Src (Y418)


Fast Activated Cell-based ELISA (FACE™) Kits provide a simple, sensitive method for detecting protein phosphorylation directly in the cell, without making extracts or performing electrophoresis and membrane blotting. These 96-well, high-throughput assays are available in both colorimetric and chemiluminescent formats for over 20 different targets (see list at right). For complete details, click the FACE™ Method tab below.

FACE c-Src Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total c-Src. The phospho-c-Src antibody recognizes c-Src when phosphorylated at Tyr418, and also recognizes Fyn and c-Yes; total-c-Src antibody recognizes c-Src regardless of the phosphorylation state. Click the c-Src Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ c-Src 1 x 96 rxns 48155 Discontinued
5 x 96 rxns 48655 Discontinued
FACE™ c-Src Chemi 1 x 96 rxns 48255 Discontinued
5 x 96 rxns 48755 Discontinued
Measurement of phosphorylated c-Src (Y418) and total c-Src using the FACE c-Src Cell Based ELISA (In Cell Western) method.

Figure 1: Measurement of phosphorylated and total c-Src.
A-431 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 100 ng/ml EGF for 20 minutes and fixed. Total and phospho c-Src were each assayed in triplicate using the phospho and total c-Src antibodies included in the FACE c-Src Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-c-Src antibody is specific for phosphorylated c-Src and was raised against a synthetic phospho-peptide corresponding to residues surrounding Tyr418 of human c-Src. This antibody recognizes c-Src when phosphorylated at this site and also recognizes Fyn and c-Yes. The total-c-Src antibody recognizes c-Src proteins regardless of the phosphorylation state.

c-Src Overview

c-Src (proto-oncogene tyrosine-protein kinase Src) is a 60 kDa tyrosine protein kinase that contains both SH2 and SH3 domains. c-Src is a lipid-modified signaling molecule (myristoylated) involved in cell-cell interactions, cell migration and proliferation through the phosphorylation of many substrates. c-Src is expressed at high levels in differentiated cells including neurons, platelets and macrophages, and can be activated by phospholipase D, insulin-like growth factor receptor, EGF-receptor, fibroblast growth factor receptor and prolactin receptor binding. c-Src can be phosphorylated on multiple sites to interact with polyoma virus middle T antigen, PYK2, HSP72 and caveolin, and efficient study methods are in high demand.

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。

Flow chart of the FACE in cell Western method that uses a cell based ELISA to measure the levels of the native and phospho forms of signaling proteins and kinases that are activated by phosphorylation.

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.