Phospho-c-Jun (Ser63) cellular kit
Sensitive and robust assay to detect and study c-Jun
This HTRF kit enables both SAPK/JNK and p38 MAPK pathway readout, measuring ATF2 modulation phosphorylated at Threonine-71.
ATF2 (Activating transcription factor 2) is a transcription factor activated by phosphorylation on threonin residues by JNK/SAPK. The phospho-ATF2 (Thr71) kit enables the quantitative detection of transcription factor ATF2 modulation phosphorylated at Threonine-71. ATF2 is also involved in PI3K/AKT, TNF, and estrogen signaling along with insulin secretion, thyroid hormone release, substance dependence, viral carcinogenesis and infectious diseases. The simple add-and-read protocol features no wash steps for a faster analysis in biological applications like oncology, diabetes or inflammation.
Mouse NIH3T3 cells were seeded at different cell densities in 96-well microplates, then stimulated with increasing concentrations of anisomycin for 30 minutes. Following the 2-plate assay protocol, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-ATF2 (Thr71) detection reagents. The HTRF signal was recorded after an overnight incubation.
Human HEK293A suspension cells were seeded at 100,000 cells/well in a 96 well half area plate, and incubated for 24h at 37°C, 5% CO2. Then cells were stimulated with different concentrations of anisomycin for 15, 30 & 60 minutes. After lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-ATF2 (Thr71) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
The human NIH3T3 cell line was seeded in a T175 flask and incubated a 37°C, 5% CO2 until confluency.
Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-ATF2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF Phospho-ATF2 T71 assay, 3,000 cells/well were sufficient to detect a signal while 6,125 cells were needed using Western Blot with an ECL detection. These results demonstrate that the HTRF phospho-ATF2 assay is 2 times more sensitive than the Western Blot.
In response to cellular stress, such as exposure to genotoxic agents, cytokines, or UV irradiation, SAPK/JNK and p38 MAP kinases are activated and phosphorylate the transcription factor ATF-2 on residues Thr69 and Thr71. Once in the nucleus, phospho-ATF2 binds to cAMP response element (CRE) or to Activator Protein-1 (AP-1) response elements, and regulates the transcription of genes involved in cell growth, survival, and DNA damage response.
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
Physiologically relevant results fo fast flowing research - Flyers
Do all cell-based kinase assays perform similarly? - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Unmatched ease of use, sensitivity and specificity assays - Videos
Get the brochure about technology comparison. - Brochures
A solution for phospho-protein analysis in metabolic disorders - Posters
A fun video introducing you to phosphorylation assays with HTRF - Videos