in-cell Western analysis for phospho JAK1 (Y1022/Y1023)


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 JAK1 Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total JAK1 (Janus Kinase 1). The phospho-JAK1 antibody recognizes JAK1 only when phosphorylated at Tyr1022 and Tyr1023. The total-JAK1 antibody recognizes JAK1 regardless of its phosphorylation state. Click the JAK1 Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ JAK1 1 x 96 rxns 48185 Discontinued
5 x 96 rxns 48685 Discontinued
FACE™ JAK1 Chemi 1 x 96 rxns 48285 Discontinued
5 x 96 rxns 48785 Discontinued
Measurement of phosphorylated JAK1 (Y1022/Y1023) and total JAK1 using the FACE JAK1 Cell Based ELISA (In Cell Western) method.

Figure 1: Measurement of phosphorylated and total JAK1.
3T3-L1 cells were cultured in 96-well plates for 48 hours and converted to adipocytes. Cells were then serum-starved for 16 hours, treated with 20 ng/ml LIF for 10 minutes and fixed. Total and phospho JAK1 were each assayed in triplicate using the phospho and total JAK1 antibodies included in the FACE JAK1 Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-JAK1 antibody is specific for phosphorylated JAK1 and was raised against a synthetic phospho-peptide corresponding to residues surrounding Tyr1022 and Tyr1023 of human JAK1. This antibody recognizes JAK1 when phosphorylated at these sites and does not cross-react with related kinases. The total-JAK1 antibody recognizes JAK1 proteins regardless of the phosphorylation state.

JAK1 Overview

The Janus family of tyrosine kinases (JAK) includes JAK1, JAK2, JAK3 and Tyk2. This family associates with a variety of cytokine receptors and is activated by ligand binding. Upon ligand binding to cytokine receptors, JAKs phosphorylate themselves and their associated receptors. This provides multiple binding sites for signaling proteins containing SH2 or other phospho-tyrosine-binding domains, including STATs, Shc, insulin receptor substrates and FAK. Thus, JAK kinases provide important links between cytokine/hormone receptors and downstream effector proteins, 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.