Phospho-c-Jun (Ser63) Cellular Assay Kit

Homogeneous HTRF cellular assay kit for detecting and studying, directly in cells, phosphorylated c-Jun at Ser63

Based on our homogeneous and robust HTRF technology, the Phospho-c-Jun assay kit is designed for detecting and studying activated c-Jun when phosphorylated at Ser63 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, c-Jun is phosphorylated on Ser63 and after the lysis of the cell membrane, phospho-c-Jun can be detected using the kit reagents. The assay is based on a sandwich immunoassay, involving two monoclonal antibodies: an anti-c-Jun antibody labelled with Eu3+-cryptate and an anti-phospho-c-Jun antibody labelled with d2. These antibodies may be pre-mixed and added in a single dispensing step.

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 c-Jun by HTRF reagents. This protocol enables the cells  viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated c-Jun 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 belongs to the mammalian Jun protein family that include also JunB and JunD. Jun proteins are bZIP (basic region-leucine zipper) transcription factors which have to dimerize to bind to their target DNA sites. c-Jun is a major component of the Activator Protein 1 (AP-1) complex, which consists of dimers of Jun with Fos and ATF proteins. AP-1 stimulation is mediated, in part, by phosphorylation of c-Jun at Ser63 and Ser73 through JNK. Once phosphorylated, c-Jun can dimerize and bind to the DNA sequence to transcript. JNK/c-Jun signaling cascade is principally activated by inflammatory cytokines and environmental stresses, but also by growth factors and GPCR agonists. c-Jun regulates numerous cellular responses including proliferation, malignant transformation, differentiation and apoptosis.

Product Performance

1. Western Blot versus HTRF assay

HEK293 cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. Stimulation was done with 500nM anisomycin for 30 min. After removal of cell culture medium, 3mL of supplemented lysis buffer 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. 160,000 cells corresponds to approximately 28µg of total protein.The HTRF assay is 8-fold more sensitive than the western-blot : 2500 cells could be detected using HTRF Phospho-c-Jun assay, while 20,000 cells were needed for the Western Blot.

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

Murine NIH-3T3 cells (50,000 cells/well) were stimulated for 30 minutes at 37°C with various concentrations of anisomycin. After a 30 minute lysis incubation time, phosphorylated c-Jun was measured using the two-plate assay protocol of the Phospho-c-Jun assay.

3. Inhibition effect of c-Jun peptide (33-57) on HEK293 stimulated with anisomycin

HEK293 cells (50,000 cells/well) were incubated for 1h at 37°C with various concentrations of cell-permeable c-Jun peptide. 50nM anisomycin (agonist) were then added and incubated for 30 minutes. After a 30 minute lysis incubation time, inhibition of c-Jun phosphorylation was measured using the Phospho-c-Jun assay. Assay was done using the two-plate assay protocol.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
cJun phospho-S63 kit - 500 tests64JUNPEG
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cJun phospho-S63 kit - 10,000 tests64JUNPEH
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cJun phospho-S63 kit - 50,000 tests64JUNPEY
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Companion products

DescriptionCat. noProduct insertMSDS
cJun phospho-S63 kit control lysate64JUNTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF

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