in-cell Western analysis for phospho ATF-2 (T71)


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 ATF-2 Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total ATF-2. The phospho-ATF-2 antibody recognizes ATF-2 when dually phosphorylated at Thr69/71 as well as when singly phosphorylated at Thr71; the total-ATF-2 antibody recognizes ATF-2 regardless of the phosphorylation state. Click the ATF-2 Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ ATF-2 1 x 96 rxns 48115 Discontinued
5 x 96 rxns 48615 Discontinued
FACE™ ATF-2 Chemi 1 x 96 rxns 48215 Discontinued
5 x 96 rxns 48715 Discontinued
Measurement of phosphorylated ATF-2 and total ATF-2 using the FACE ATF-2 Cell Based ELISA (In Cell Western) method.

Figure 1: Measurement of phosphorylated and total ATF-2.
NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 25 µg/ml of anisomycin for 30 minutes and fixed. Total and phospho ATF-2 were each assayed in triplicate using the phospho and total ATF-2 antibodies included in the FACE ATF-2 Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-ATF-2 antibody is specific for phosphorylated ATF-2 and was raised against a synthetic phospho-peptide corresponding to residues surrounding Thr71 of human ATF-2. This antibody recognizes ATF-2 when phosphorylated at this site and does not cross-react with c-Jun, CREB or other transcription factors. This antibody recognizes both ATF-2 when dually phosphorylated at Thr69/71 as well as ATF-2 when singly phosphorylated at Thr71. The total-ATF-2 antibody recognizes ATF-2 proteins regardless of the phosphorylation state.

ATF-2 Overview

ATF-2 is a member of the ATF/CREB family of leucine zipper DNA binding proteins that regulate transcription. This family of transcription factors play an important role in cell growth and differentiation, as well as in stress and immune responses. ATF-2 is a nuclear protein that binds to DNA as a dimer, and can form both homo- and heterodimers with members of the ATF/CREB and Jun/Fos families. The ATF-2/c-Jun heterodimer acts as a stronger activator than the ATF-2 homodimer. ATF-2 plays a role in c-Jun-dependent cell cycle progression, cell survival and apoptosis, making efficient study methods 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.