Cellomics HCS Reagent Kits for multiplex analysis of NFκB and c-Jun.

Figure 1. Staining of phospho-c-jun (A, C) and NFkB (B, D) in HeLa cells treated with media only (A, B) or with 25 ng/ml TNF-a for 25 min (C, D). Proteins were detected according to the kit protocol. The same cells are shown in panels A and B, and in C and D. The cells were imaged using a Cellomics ArrayScan HCS Reader.

Description

Nuclear factor kappa B (NFκB) and c-jun are transcription factors important for many physiological processes and responses such as cell proliferation, cell survival, cellular responses to stress and immune response. Normally, NFκB is present in the cytoplasm as a complex. Upon activation by cytokines, such as tumor necrosis factor alpha (TNF-a). NFκB is translocation into the nucleus, where it can bind to specific DNA sequences and activate gene transcription. NFκB must be in the nucleus to induce specific gene expression. Thus, as with many other transcription factors, translocation from the cytoplasm to the nucleus is a definitive measure of NFκB activation.

TNF-a, in addition to activating the NFκB-mediated signaling pathway, can activate c-jun. c-Jun is activated through phosphorylation in the activation domain by Jun-N-terminal kinases (JNKs). Phosphorylated Jun family members then form homodimers or heterodimeric complexes with Fos, composing the AP-1 transcription factor, which migrates into the nucleus. The AP-1 transcription factor has been implicated in stress-induced apoptosis, heat shock, T-cell activation, cellular responses to inflammatory cytokines and cellular transformation, as well as ischemia/reperfusion.

The Multiplexed NFkB and c-Jun Activation HCS Reagent Kits provide a highly effective tool for the simultaneous quantification of NFkB and c‑jun activation in the same cell (Figure 1). These kits allow direct measurements of NFkB and phospho-c-jun translocation from the cytoplasm to the nucleus using a fixed end-point assay based on immunofluorescence. The primary antibodies are specific for their targets and have minimal cross-reactivity with other targets. Prepared cells can be analyzed using standard fluorescence microscopy or using a Cellomics ArrayScan HCS Reader with the Molecular Translocation BioApplication, affording fully automated plate handling, focusing, image acquisition, analysis, quantification, and data storage.

Features

• Available in multiplex or singleplex configurations
• Optimized protocol requiring no cell lysis, purification or filtration steps
• Immunofluorescence-based assay provides sensitive results without radioactivity
• Cells prepared with the HCS Reagent Kits may be analyzed directly via standard fluorescence microscopy; or HCS Imaging Systems including the ArrayScan HCS Reader
• Primary antibodies are specific for their targets and have minimal cross-reactivity with other targets

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

• NFkB primary antibody
• c-Jun primary antibody
• DyLight™ 549 conjugated goat anti-rabbit IgG
• Alexa Fluor^® 488 conjugated goat anti-mouse IgG
• Hoechst dye
• Wash buffer (10X Dulbecco's PBS)
• Permeabilization buffer (10X Dulbecco's PBS with 1% Triton^® X-100)
• Blocking buffer (10X)
• Thin plate seals

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:
Phospho-c-Jun Kit
Phospho-c-Jun and Phospho-JNK Multiplex Kit

See all Cellomics HCS Reagent Kits