NFkB and c-Jun Activation Kits

• 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 System including the ArrayScan HCS Reader
• Primary antibodies are specific for their targets and have minimal cross-reactivity with other targets    

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 detection in cells grown on standard high-density microplates. The primary antibodies are specific for their targets and have minimal cross-reactivity with other targets.

The orange NFkB and green c-jun duplex probes kits contain primary antibodies towards NFkB and phosphorylated c-jun (rabbit polyclonal and mouse monoclonal, respectively) and secondary antibodies conjugated with orange and green fluorophores (DyLight™ 549 Goat Anti-rabbit and DyLight 488 Goat Anti-mouse Antibodies, respectively). The orange NFkB probe kits contain a rabbit polyclonal primary antibody towards NFkB and a DyLight 549-conjugated Secondary Antibody.

The Multiplexed NFkB and c-Jun Activation Kits have been optimized for use with the Thermo Scientific Cellomics ArrayScan® HCS Reader using the Molecular Translocation BioApplication Software Module. These kits can be used with other Cellomics BioApplications. Thus, automated plate handling, focusing, cell image acquisition/processing, and data analysis/management are combined in one high-content screening (HCS) system to assay for test compounds regulating NFkB and c-jun activation. In addition to HCS instruments, cells labeled by the reagents in this kit can be viewed and analyzed by other fluorescence microscopy-based instruments.

Figure 1. Staining of phospho-c-Jun (A, C) and NFκB (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.

Figure 2. Dose Response of NFkB and Phospho-c-Jun activation by TNF-alpha.

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