The phospho-JNK (Thr183/Tyr185) kit enables the cell-based quantitative detection of JNK protein phosphorylated at Thr183/Tyr185.
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  • Faster and more convenient than ELISA
  • Ready-to-use
The phospho-JNK (Thr183/Tyr185) kit enables the cell-based quantitative detection of JNK protein phosphorylated at Thr183/Tyr185.
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Overview

Based on our homogeneous and robust HTRF technology, the Phospho-JNK assay kit is designed for detecting and studying activated JNK when phosphorylated at Thr183 and Tyr185 directly in whole cells. Using a streamlined mix and read, no wash protocol, this kit can be used from basic research to High Throughput drug screening.

Phospho-JNK (Thr183/Tyr185) assay principle

The phospho-JNK (Thr183/Tyr185) assay measures JNK when phosphorylated at Thr183/Tyr185. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The phospho-JNK (Thr183/Tyr185) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-JNK (Thr183/Tyr185) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding phospho-JNK (Thr183/Tyr185) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-JNK (Thr183/Tyr185) 1-plate assay protocol

Detection of Phosphorylated JNK (Thr183/Tyr185) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

HTRF phospho-JNK assay compared to Western Blot

HEK293 cells were cultured for 2 days. Stimulation was done with 500nM anisomycin for 30 min. Following cell lysis, soluble supernatants were collected by centrifuging for 10 minutes. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. 100,000 cells corresponds to 16 µg of total protein. HTRF phospho-JNK assay shows the same level of sensitivity as Western Blot: 3,125 cells were needed for the two technologies.

Anysomycin dose-response on NIH-3T3 cells

Murine NIH-3T3 cells (100,000 cells/well) were stimulated for 30 minutess at 37°C with various concentrations of anisomycin. After a 30 minutes lysis incubation time, phosphorylated JNK was measured using the two-plate assay protocol of the Phospho-JNK assay

Inhibition effect of IL-1Ra on Jurkat cells stimulated with IL-1ß

Suspended Jurkat cells (80,000 cells/well) were incubated for 10 min at 37°C with various concentrations of IL-1Ra (IL-1 receptor antagonist). 10nM IL-1ß (agonist) were then added and incubated for 10 minutes. After a 30 minutes lysis incubation time, inhibition of JNK phosphorylation was measured using the Phospho-JNK assay. The asssay was done using the one-plate assay protocol.

Simplified Pathway for Phospho-JNK (Thr183/Tyr185) assay kit

C-Jun N-terminal kinases (JNKs), also called Stress-activated protein kinases (SAPKs) are members of the MAPK family. JNKs are activated by a variety of environmental stresses, inflammatory cytokines and, in some instances, by growth factors and GPCR agonists. Activated JNK translocates to the nucleus where it can phosphorylate multiple transcription factors, such as c-Jun, ATF-2 and p53. The JNK pathway regulates numerous cellular responses including proliferation, survival & apoptosis, neural development, inflammation, metabolism and DNA repair.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

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