TF-Detect™ c-Jun activity assay

Supplier
GeneCopoeia
Cat. no.
TAK-JUN-196
Kit size
96 rxns
Links
Protocol

Features

• High sensitivity: Detect Ser73-phosphorylated c-Jun in as low as 0.2 µg of nuclear lysate
• Multiple species: Detects human, mouse or rat active c-Jun
• 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

TF-Detect™ Human c-Jun activity assay kit enables fast and sensitive detection and quantification of Ser73-phosphorylated in a 96-well format. Double-stranded oligonucleotides containing a c-Jun consensus binding site are immobilized in a 96-well plate. The c-Jun proteins present in nuclear extracts are captured by the immobilized oligonucleotides specifically and then detected by a phospho 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.

Background of AP-1/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.

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.