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FACE™ c-Jun

in-cell Western analysis for phospho c-Jun (S63) or (S73)

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.

Each FACE c-Jun Kit provides 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total c-Jun. The S63 kit contains phospho-c-Jun antibody that recognizes c-Jun only when phosphorylated at Ser63; it does not recognize the phosphorylated forms of JunD or JunB. The S73 kit contains phospho-c-Jun antibody that recognizes c-Jun when phosphorylated at Ser73, but it also recognizes JunD when phosphorylated at Ser100. Both kits contain a total-c-Jun antibody that recognizes c-Jun regardless of the phosphorylation state. Click the c-Jun Info tab below for data and more information.

 
Name Format Cat No. 価格 (税抜)  
FACE™ c-Jun (S63) 1 x 96 rxns 48125 ¥97,000 Buy Now
5 x 96 rxns 48625 ¥389,000 Buy Now
FACE™ c-Jun (S63) Chemi 1 x 96 rxns 48225 ¥106,000 Buy Now
5 x 96 rxns 48725 ¥427,000 Buy Now
FACE™ c-Jun (S73) 1 x 96 rxns 48135 ¥97,000 Buy Now
5 x 96 rxns 48635 ¥389,000 Buy Now
FACE™ c-Jun (S73) Chemi 1 x 96 rxns 48235 ¥106,000 Buy Now
5 x 96 rxns 48735 ¥427,000 Buy Now
Measurement of phosphorylated c-Jun (S63), phosphorylated c-Jun (S73) and total c-Jun using the FACE c-Jun (S63) and FACE c-Jun (S73) Cell Based ELISAs (In Cell Westerns).
 
Figure 1: Measurement of phosphorylated and total c-Jun.

NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 25 µg/ml of anisomycin for 30 minutes and fixed. Total and phospho c-Jun were each assayed in triplicate using either the phospho-c-Jun (S63), phospho-c-Jun (S73) or total c-Jun antibodies included in the FACE c-Jun Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-c-Jun (S63) antibody is specific for phosphorylated c-Jun (S63) and was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 63 of human c-Jun. This antibody recognizes c-Jun when phosphorylated at this site and does not recognize the phosphorylated forms of JunD or JunB. The phospho-c-Jun (S73) antibody is specific for phosphorylated c-Jun (S73) and was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 73 of human c-Jun. This antibody recognizes c-Jun only when phosphorylated at this site and also recognizes JunD when phosphorylated at Serine 100. The total-c-Jun antibody recognizes c-Jun proteins regardless of the phosphorylation state. Each antibody is provided in a quantity sufficient for one 96-well assay.

c-Jun Overview

c-Jun is a component of the transcription factor AP-1, which binds and activates transcription at TRE/AP-1 elements. The c-Jun oncogene encodes a nuclear protein, p39, that interacts with the AP-1 c-Fos oncogene product and forms a transacting heterodimer. c-Jun plays an important role in the regulation of gene expression and signal transduction processes and is also suggested to be actively involved in the differentiation of B-chronic lymphocytic leukemia cells and Hodgkin’s Reed-Sternberg cells as well as in the malignant transformation of melanocytes, which makes it a promising drug target.

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.