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TF-Detect™ AP-1/c-Jun Activity Assay Kit

Introduction

c-Jun, together with Fos and other Jun-related proteins, forms a homodimeric or heterodimeric transcription factor complex AP-1. TF-Detect™ AP-1/c-Jun Activity Assay Kit enables fast and sensitive detection of Ser73-phosphorylated c-Jun in human, mouse or rat samples in a 96-well format. Double-stranded oligonucleotides containing the AP-1/c-Jun consensus binding site are immobilized in the 96-well plate. The c-Jun proteins present in nuclear extracts are captured by the immobilized oligonucleotides specifically. The Ser73-phosphorylated c-Jun is detected by a phosphor-c-Jun antibody and a HRP-conjugated secondary antibody. The colorimetric signal generated by HRP substrate TMB can be easily quantified by spectrophotometry. The Ser73 phospho-c-Jun antibody that detects Ser73-phosphorylated c-Jun also recognizes Ser100-phosphorylated JunD, as this site is conserved between c-Jun and JunD.

 

Advantages

Multiple species

  • Detects human, mouse or rat active c-Jun

Sensitive

  • Detect Ser73-phosphorylated c-Jun in as low as 0.2 µg of nuclear lysate

HTS compatible

  • Optimized 96-well format for high-throughput analysis on 96-well plate readers
  • Single strip (8-well) assay can also be performed

Fast

  • 3 and 1/2 hours from preparation to detection

 

Background of c-Jun

c-Jun, together with Fos and other Jun-related proteins, forms a homodimeric or heterodimeric transcription factor complex AP-1. AP-1 binds to a TPA DNA Response Element (TRE), the heptamer enhancer motif 5'-TGA[C/G]TCA-3', and regulates gene expression in response to a variety of stimuli including growth factors, tumor promoters, and cytokines. AP-1 controls cellular processes including differentiation, proliferation, apoptosis and stress response.

c-Jun is tightly regulated post-translationally through phosphorylation. It has several phosphorylation sites. The C-terminal one is near the DNA binding domain of c-Jun and its phosphorylation represses c-Jun activity during the resting condition. The other two phosphorylation sites, Ser63 and Ser73, are near the transactivation domain of c-Jun. SAPK/JNKs activate c-Jun by phosphorylating Ser63 and Ser73.

Protocol and Data

Protocol Overview

 

 

 

Nuclear-Lysate

Figure 1. The activities of c-Jun proteins in the nuclear extracts of MCF-7 (left) and NIH3T3 (right) cells were detected using the TF-Detect AP-1/c-Jun Activity Assay Kit. Both cell types were treated with UV light for 20 Sec before harvesting. The cellular nuclear extracts were prepared following the Preparation of Nuclear Extract protocol in the Appendix.

To Order

Buy Catalog# Product Description Price
TF002 TF-Detect™ AP-1/c-Jun Activity Assay Kit (one 96-well plate) Quick detection of active c-Jun in human, mouse or rat cellular nuclear extracts $545

   

 

 

 

 

 

 

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