Phospho-JNK (Thr183 / Tyr185) Cellular Assay Kit

Homogeneous HTRF cellular assay kit for detecting and studying, directly in cells, phosphorylated JNK at Thr183 and Tyr185

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.

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

The assay can be run with cell lysates or using whole cells.

Upon activation, JNK is phosphorylated on Thr183 and Tyr185 and after the lysis of the cell membrane, phospho-JNK can be detected using the kit reagents. The assay is based on a sandwich immunoassay, involving two monoclonal antibodies: an anti-phospho-JNK antibody labelled with Eu3+-cryptate and an anti-JNK antibody labelled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.

The assay can be run under a two-plate protocol. It can also be further streamlined to a one-step assay.

Two-plate assay protocol

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated JNK by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated JNK with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, designed for HTS, enables miniaturization while maintaining a high quality output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

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. JNK proteins are encoded by three genes, JNK1, JNK2 and JNK3, which undergo alternate splicing to generate several isoforms for each gene. Specific stimuli trigger the activation of MAPKKKs, which then phosphorylate the MAPKKs MKK4 and MKK7, which in turn activate SAPK/JNK by phosphorylation of Thr183 and Tyr185. Activated JNK translocates to the nucleus where it can phosphorylate multiple transcription factors, such as c-Jun, ATF-2 and p53. JNK pathway regulates numerous cellular responses including proliferation, survival & apoptosis, neural development, inflammation, metabolism and DNA repair.

Product Performance

1. Western Blot versus HTRF assay

HEK293 cells were grown in a T175 flask at 37°C, 5% CO2 for2 days.

Stimulation was done with 500nM anisomycin for 30 min. After removal of cell culture medium, 3mL of supplemented lysis buffer # 3 were added and incubated for 30 min.

Soluble supernatants were collected after 10 min centrifuging. 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: 3125 cells were needed for the two technologies.

2. Anysomycin dose-response on NIH-3T3 cells

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

3. Inhibition effect of IL-1Ra on Jurkat cells stimulated with IL-1β

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-1β (agonist) were then added and incubated for 10 minutes. After a 30 minute lysis incubation time, inhibition of JNK phosphorylation was measured using the Phospho-JNK assay. The asssay was done using the one-plate assay protocol.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
JNK phospho-T183/Y185 kit - 500 tests64JNKPEG
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JNK phospho-T183/Y185 kit - 10,000 tests64JNKPEH
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JNK phospho-T183/Y185 kit - 50,000 tests64JNKPEY
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Companion products

DescriptionCat. noProduct insertMSDS
JNK phospho-T183/Y185 kit control lysate64JNKTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #3 - 130 mL64KL3FDF

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