in-cell Western analysis for phospho STAT4 (Y693)


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 STAT4 Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total STAT4. The phospho-STAT4 antibody recognizes STAT4 only when phosphorylated at Tyr693, and does not cross-react with other sites. The total-STAT4 antibody recognizes STAT4 regardless of its phosphorylation state. Click the STAT Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ STAT4 1 x 96 rxns 48320 Discontinued
5 x 96 rxns 48820 Discontinued
FACE™ STAT4 Chemi 1 x 96 rxns 48420 Discontinued
5 x 96 rxns 48920 Discontinued
Measurement of phosphorylated STAT2 (Y689) and total STAT2 using the FACE STAT2 Cell Based ELISA (In Cell Western) method.

Figure 1: Measurement of phosphorylated and total STAT.
NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 50 ng/ml PDGF for 5 minutes and fixed. Total and phospho STAT2, STAT4 and STAT6 were each assayed in triplicate using the phospho and total STAT antibodies included in the FACE STAT Kits. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-STAT2 antibody is specific for phosphorylated STAT2 at Tyrosine 689 and does not cross-react with other sites. The total-STAT2 antibody recognizes STAT2 proteins regardless of the phosphorylation state. The phospho-STAT4 antibody is specific for phosphorylated STAT4 at Tyrosine 693 and does not cross-react with other sites. The total-STAT4 antibody recognizes STAT4 proteins regardless of the phosphorylation state. The phospho-STAT6 antibody is specific for phosphorylated STAT6 at Tyrosine 641 and does not cross-react with other sites. The total-STAT6 antibody recognizes STAT6 proteins regardless of the phosphorylation state.

STAT Overview

Signal transducers and activators of transcription (STAT) proteins are latent transcription factors that are activated by phosphorylation via tyrosine kinases. Over 35 different extracellular polypeptides activate Janus kinase associated receptors, leading to phosphorylation of Janus kinases and the subsequent phosphorylation of STAT proteins. Upon phosphorylation, the STAT proteins dimerize and migrate to the nucleus where they exert transcriptional activation. Phosphorylation of a single tyrosine localized around residue 700 is crucial for activation of each STAT family member. STAT1 is involved in the activation of IFNα and IFNγ genes, STAT2 in the activation of IFNα genes, STAT4 and STAT6 in T-helper cell development and STAT5 in milk production. Disruption of STAT functions in mouse leads to several defects such as immune deficiency (STAT1), embryonic lethality (STAT2), lack of gastrulation (STAT3), T-helper 1 cell dysfunction (STAT4), lack of lactation (STAT5A, 5B) and T-helper 2 cell dysfunction (STAT6). The disruption of STAT signaling blocks neoplastic transformation, thus making inhibitors of STAT proteins candidates for the treatment of cancer.

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.