ATF-2 Activation Kit

• 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

The Cellomics ATF-2 Activation Kit enables quantitation of ATF-2 activation by directly measuring its translocation from the cytoplasm to the nucleus. The assay is performed on live cells growing on standard high density microplates. The kit is supplied with a phospho-ATF-2 specific antibody and a DyLight™ 488-conjugated Secondary Antibody. The nuclear region is identified by Hoechst Dye, which is also included.

The ATF-2 transcription factor is activated in response to inflammatory cytokines and cellular stress, including genotoxicity and ischemia/reperfusion. ATF-2 is activated through phosphorylation of 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 (CRE) present in the promoters of many genes, stimulating gene transcription. Activation of ATF-2 can be quantified by its translocation to the nucleus. The immunofluorescent assay uses a validated phospho-ATF-2 specific antibody that is non-reactive to other cAMP response element-binding protein (CREB) or ATF family members.

The ATF-2 Activation Kit, in combination with the Thermo Scientific ArrayScan® HCS Reader and the Cytoplasm to Nucleus Translocation or Molecular Translocation BioApplication software, enable automated plate handling, focusing, cell image acquisition, analysis and quantification of ATF-2 activation.

Figure 1. Images of immunofluorescently stained NIH 3T3 cells before (left) and after (right) activation by ATF-2 by anisomycin. Cells were stimulated with 200 ng/mL anisomycin for 30 min and then prepared using the kit protocol. Nuclei appear blue (Hoechst) and ATF-2 appears green. Images were aquired using the ArrayScan® HCS Reader.

Figure 2. Diagrammatic view of the principle of the ATF-2 Activation Reagent Kit.  ATF-2 (green), normally present in the cytoplasm, translocates to the nucleus.