in-cell Western analysis for phospho FKHR (FOXO1) (T24)

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 FKHR Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total FKHR (Forkhead). The phospho-FKHR antibody recognizes FKHR only when phosphorylated at Thr24, and also recognizes FKHRL1 phosphorylated at Thr32 and AFX phosphorylated at Thr28. The total-FKHR antibody recognizes FKHR regardless of its phosphorylation state. Click the FKHR Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ FKHR (FOXO1) 1 x 96 rxns 48160 ¥98,500 Buy
5 x 96 rxns 48660 ¥396,000 Buy
FACE™ FKHR (FOXO1) Chemi 1 x 96 rxns 48260 ¥98,000 Buy
5 x 96 rxns 48760 ¥396,000 Buy
Measurement of phosphorylated FKHR (T24) and total FKHR using the FACE FKHR Cell Based ELISA (In Cell Western) method.
Figure 1: Measurement of phosphorylated and total FKHR.

CHO-K1 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20% FBS for 30 minutes and fixed. Total and phospho FKHR were each assayed in triplicate using the phospho and total FKHR antibodies included in the FACE FKHR Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-FKHR antibody is specific for phosphorylated FKHR and was raised against a synthetic phospho-peptide corresponding to residues surrounding Thr24 of human FKHR. This antibody recognizes FKHR only when phosphorylated at Thr24 and also recognizes FKHRL1 when phosphorylated at Thr32 and AFX when phosphorylated at Thr28. The total-FKHR antibody recognizes FKHR proteins regardless of the phosphorylation state.

FKHR Overview

The Forkhead transcription factor FKHR (FOXO1) regulates expression of gluconeogenic factors, and plays a key role in the insulin regulation of glucose levels. Besides its role in glucose homeostasis, FKHR has been shown to be involved in pathological processes such as type 2 diabetes, cancer and organismal aging. The transcriptional output of FKHR is controlled by a two-tiered mechanism of phosphorylation and acetylation, and alterations in this balance can result in profound effects.

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.