in-cell Western analysis for phospho Bad (S112)


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 Bad Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total Bad. The phospho-Bad antibody recognizes Bad only when phosphorylated at Ser112; the total-Bad antibody recognizes Bad regardless of the phosphorylation state. Click the Bad Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ Bad 1 x 96 rxns 48165 Discontinued
5 x 96 rxns 48665 Discontinued
FACE™ Bad Chemi 1 x 96 rxns 48265 Discontinued
5 x 96 rxns 48765 Discontinued
Measurement of phosphorylated Bad and total Bad using the FACE Bad Cell Based ELISA (In Cell Western) method.

Figure 1: Measurement of phosphorylated and total Bad.
COS-7 cells were cultured in 96-well plates and serum-starved for 24 hours. Cells were then treated with 200 nM of PMA for 30 minutes and fixed. Total and phospho Bad were each assayed in triplicate using the phospho and total Bad antibodies included in the FACE Bad Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-Bad antibody is specific for phosphorylated Bad and was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 112 of mouse Bad. This antibody recognizes Bad only when phosphorylated at this site and does not cross-react with other sites or related family members. The total-Bad antibody recognizes Bad proteins regardless of the phosphorylation state.

Bad Overview

Bad (Bcl-2 antagonist of cell death) is a proapoptotic member of the Bcl-2 family that displaces Bax from binding to Bcl-2 and Bcl-xL, which results in cell death. Bad forms heterodimers with other Bcl-2 family members and localizes to the outer mitochondrial membrane. When phosphorylated in the presence of survival signals, Bad translocates to the cytoplasm and blocks the induction of apoptosis. In the absence of survival signals, dephosphorylated Bad is localized in the outer membrane of the mitochondria where it activates mitochondrial-mediated apoptotic pathways. Thus, Bad is a unique Bcl-2 family member that plays a key role in the transduction of upstream survival signals to the Bcl-2 apoptosis decision machinery, and high-throughput study methods for monitoring Bad phosphorylation 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.