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FACE™ MEK1/2

in-cell Western analysis for phospho MEK1/2 (S217/S221)

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 MEK1/2 Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total MEK1/2. The phospho-MEK1/2 antibody recognizes MEK1/2 only when phosphorylated at Ser217/Ser221, and also reacts with MEK1/2 when singly phosphorylated at Ser217 but not at Ser221. The total-MEK1/2 antibody recognizes MEK1/2 proteins regardless of their phosphorylation states. Click the MEK1/2 Info tab below for data and more information.

 
Name Format Cat No. 価格 (税抜)  
FACE™ MEK1/2 1 x 96 rxns 48180 ¥97,000 Buy Now
5 x 96 rxns 48680 ¥389,000 Buy Now
FACE™ MEK1/2 Chemi 1 x 96 rxns 48280 ¥106,000 Buy Now
5 x 96 rxns 48780 ¥427,000 Buy Now
Measurement of phosphorylated MEK1/2 (S217/S221) and total MEK1/2 using the FACE MEK1/2 Cell Based ELISA (In Cell Western) method.
 
Figure 1: Measurement of phosphorylated and total MEK1/2.

NIH/3T3 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 MEK1/2 were each assayed in triplicate using the phospho and total MEK1/2 antibodies included in the FACE MEK1/2 Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-MEK1/2 antibody is specific for phosphorylated MEK1/2 and was raised against a synthetic phospho-peptide corresponding to residues surrounding Ser217/Ser221 of human MEK1/2. This antibody recognizes MEK1/2 only when phosphorylated at Ser217/Ser221, and also reacts with MEK1/2 when singly phosphorylated at Ser217 but not at Ser221. It does not cross-react with related kinases. The total-MEK1/2 antibody recognizes MEK1/2 proteins regardless of their phosphorylation states.

MEK1/2 Overview

MEK1 and MEK2, also called MAP kinase kinase and ERK activator kinase, are dual-specificity protein kinases that function in a Mitogen Activated Protein Kinase (MAPK) cascade controlling both cell growth and differentiation. MEK1/2 is activated by a wide variety of growth factors and cytokines, and also by membrane depolarization and calcium influx, with the predominant activators being Raf kinases. MEK is involved in the MAPK signaling pathway of Ras-Raf-MEK-ERK. This signaling cascade is an important target in cancer therapy, making high-throughput study methods in 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。

 
FACE flow chart
 
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.