The Phospho-c-Jun (Ser63) kit enables the cell-based quantitative detection of cJun protein phosphorylated at Ser63.

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  • Ease-of-use
  • High sensitivity

The Phospho-c-Jun (Ser63) kit enables the cell-based quantitative detection of cJun protein phosphorylated at Ser63.

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The Phospho-c-Jun (Ser63) Cellular Assay kit is designed for the robust quantification of phoshorylated c-Jun at Ser-36 directly in cell lysates. Using a streamlined mix-and-read no-wash protocol, this kit can be used from basic research to High Throughput drug screening.

Phospho-c-Jun (Ser63) assay principle

The Phospho-c-Jun (Ser63) assay measures c-Jun when phosphorylated at Ser63. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-c-Jun (Ser63) 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-c-Jun (Ser63) 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-c-Jun (Ser63) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-c-Jun (Ser63) 1-plate assay protocol

Detection of Phosphorylated c-Jun (Ser63) 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.

WB versus HTRF assay for Phoshpo-c-Jun (Ser63) kit

HEK293 cells were cultured for two days and stimulated with 500nM anisomycin for 30 min. After removal of cell culture medium, lysis buffer was added and the cells were incubated for 30 min. Supernatants were collected following centrifugation and equal amounts of lysates were used for a side by side comparison of WB and HTRF. The HTRF assay proved to be 8-fold more sensitive than the western-blot assay.

Anysomycin dose-response on NIH-3T3 cells for Phospho-c-Jun kit

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

Inhibition effect of c-Jun 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 minutess. After a 30 minutes 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.

Simplified pathway of c-Jun protein family

c-Jun belongs to the mammalian Jun protein family which are transcriptoin factors that have to dimerize to bind to their target DNA sites. c-Jun is a major component of the Activator Protein 1 (AP-1) complex. Phosphorylation of c-Jun at Ser63 and Ser73 through JNK stimulates AP-1. Once phosphorylated, c-Jun can dimerize and bind to DNA sequences and affect transription. c-Jun regulates numerous cellular responses including proliferation, malignant transformation, differentiation and apoptosis.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

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

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® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

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

Product Insert cJun P-S63 Kit / 64JUNPEG-64JUNPEH

64JUNPEG-64JUNPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides