Cellomics HCS Reagent Kit for Activating Transcription Factor 2 (ATF-2).

Description

The Activating Transcription Factor-2 (ATF-2) Activation HCS Reagent Kit provides high-content screening (HCS)-quality fluorescence reagents and a validated, optimized protocol for measuring and visualizing the activation of the ATF-2 transcription factor. Responding to the inflammatory cytokines and cellular stressors including genotoxicity and ischemia/reperfusion, ATF-2 is activated through threonines 69 and 71 by members of the SAPK family. Once activated, ATF-2 forms complexes with Jun family or other ATF family members. These complexes bind to the cAMP Response Element found in the promoters of many genes, stimulating gene transcription. Because ATF-2 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, ATF-2 translocation represents a functional, more biologically relevant assay in comparison to gel mobility shift assays.

With this cell based assay kit, activation of the ATF-2 is quantified by its translocation to the nucleus (Figure 1). This fixed end-point assay is based on immunofluorescence, using an ATF-2 specific antibody that has been validated for non cross-reactivity to other CREB or ATF family members. Inhibitors of ATF-2 translocation are screened by stimulating live cells with the positive control compound (included in the kit) following test compound treatment. Replacing the positive Control Compound with the test compound identifies agonists of ATF-2 translocation. Following test compound incubation, the cells are fixed, permeabilized, labeled with a primary antibody to ATF-2, and stained with a fluorescently-labeled secondary antibody (Figure 1). Prepared cells can be analyzed by using standard fluorescence microscopy or using a Cellomics fully automated HCS Readers and the Cytoplasm to Nucleus Translocation BioApplication, affording automated plate handling, focusing, image acquisition, analysis, and quantification of ATF-2 activation.

Features

• Cell-based assay provides an earlier and more definitive measure of ATF-2 activation relative to electophoretic mobility shift or gene reporter assays
• Cells prepared with the HCS Reagent Kits may be analyzed directly via standard fluorescence microscopy; or any HCS Imaging System.
• Prepared cells are ready for analysis in less than 3 hours post-test compound incubation; kit protocol requires no cell lysis, purification or filtration steps
• Immunofluorescence-based screening assay provides sensitive quantitative and qualitative results without radioactivity
• Validated protocol included with all necessary stains and components

Related Poster Presentation

Amy M. Peters and Jeffrey R. Haskins (2005) Cell Type Specificity for ATF-2 Activation.

Kit Contents

• ATF-2 primary antibody
• Alexa Fluor® 488-conjugated secondary antibody
• Hoechst dye
• Positive control compound
• Wash buffer (10x)
• Permeabilization buffer (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).

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