Cellomics HCS Reagent Kit for activated (phosphorylated) transcription factor c-Jun.

Description

c-Jun is activated through phosphorylation by c-Jun-N-terminal kinases (JNKs) Phosphorylated c-Jun family members then homodimerize of form a heterodimeric complex with c-Fos creating Activator Protein (AP) transcription factor, which migrates into the nucleus. AP-1 has been implicated in stress-induced apoptosis, heat shock, T-cell activation, cellular responses to inflammatory cytokines, and cellular transformation (Davis, RJ. Biochem. Soc. Symp. 64:1-12), as well as ischemia/reperfusion (Morooka, H, et al. J. Biol. Chem. 270: 30084-30092). c-Jun is activated by a wide variety of stimuli, including anisomycin, epidermal growth factor, hydrogen peroxide, and TNF-alpha.

The c-Jun Activation HCS Reagent Kit provides High Content Screening (HCS)-quality fluorescence reagents and a validated, optimized protocol for the measurement and visualization of the activation of the c-Jun transcription factor. The assay is performed on live cells growing on standard high-density microplates and is based on immunofluorescence, employing an antibody specific for phosphorylated c-Jun. This antibody has been validated to verify non-cross-reactivity to other c-Jun or c-Fos family member. The optimized protocol included with the c-Jun Activation Kit is for a fixed end-point assay. Inhibitors of c-Jun translocation are screened by stimulating cells with a control inducer such as anisomycin following the exposure of live cells to the test compounds. Replacing anisomycin in the assay with the test compounds identifies agonists of c-Jun translocation. Translocation can be quantified as the difference or ration in cytoplasmic to nuclear intensity of the labeled transcription factor (Figures 1&2).

Because c-Jun must be in the nucleus to induce gene expression, its translocation is a definitive measure of its activation, and marks an earlier event than reporter gene expression. Further, c-Jun translocation represents a functional, more biologically relevant assay in comparison to gel mobility shift assays and does not require additional isolation steps. Prepared cells can be analyzed using standard fluorescence microscopy or using a Cellomics fully automated HCS Reader with the Cytoplasm to Nucleus Translocation BioApplication, affording automated plate handling, focusing, image acquisition, analysis, and quantification and data storage.

Features

• Quantify endogenous c-Jun activation specifically and reliably in whole cells
• Immunofluorescence-based screening assay provides sensitive quantitative and qualitativeresults without radioactivity.
• No cell lysis, purification, or filtration steps
• No need to generate stable clonal lines; entire assay completed in 3 h (post-stimulation)
• Earlier and more definitive measure of transcription factor activation relative to electophoretic mobility shift or gene reporter assay
  

Related Poster Presentations

	Kay Opperman, Eric Hommema, Brian Webb, and Richik N. Ghosh (2008) Combining Multiplexed, High-Content Analysis with Antibody Arrays to Monitor and Correlate Intracellular and Extracellular Inflammatory Responses.
	Ulla Henriksen, Kay Opperman, Jacob Fog, Richik N. Ghosh, Frosty Loechel, Morten Praestegaard (2007) Profiling of Multiple Signal Pathway Activities by Multiplexing Antibody and GFP-based Translocation Assays.
	Ulla Henriksen, Kay Opperman, Jacob Fog, Richik N. Ghosh, Frosty Loechel, Morten Praestegaard (2007) Multiplexing of Antibody and GFP-based Translocation Assays: A Pathway to Better Profiling of Cellular Signalling.
	Jason Quarles, Erica Webber, Megan Cuda, Jeff Haskins, Greg Endress (2004) Automated Dual Antibody High-Content Screening on the BioCube System.
	Chandrasekaran Vasudevan, Patrick Maunder, Jeffrey Haskins, Natalie Fursov Mei Cong and Zhong Zhong (2004) The Use of Division-Arrested Cells Improves the Robustness of High Content Screening Assays.
	Yih-Tai Chen, Kenneth A. Guiliano, and Jeffrey R. Haskins (2003) High-Content Screening of Gene Knock-downs Induced with siRNA Transfection.
	Chandrasekaran Vasudevan, Oleg Lapets and Jeffrey R. Haskins (2003) Simultaneous and Quantitative Analysis of Signal Transduction Pathways with a Cell-Based High Content Screening Application.
	Yih-Tai Chen, Kenneth A. Giuliano, and Jeffrey R. Haskins (2003) Validation of siRNA Gene Knockdown Through Automated Phenotypic Analysis of Cellular Function.

Kit Contents

• phospho-c-Jun primary antibody
• Alexa Fluor® 488—conjugated secondary antibody
• Hoechst dye
• Positive control compound
• Wash buffer (10x)
• Permeabilization buffer (10x)
• Detergent buffer (10x)
• Positive control compound (Anisomycin) (10x)
• Plate seals (10x)

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.

Choose "Order Online" in table to see pricing and order online (U.S. only).

See also:
NFkB and Phospho-c-Jun Multiplex Kit
Phospho-c-Jun and Phospho-JNK Multiplex Kit

See all Cellomics HCS Reagent Kits