Cellomics HCS Reagent Kits for multiplex analysis of HIF-1 alpha, Phospho-CREB and FOXO3a.

Description

These Transcription Factor Activation HCS Reagent Kits are for the simultaneous quantitation of HIF-1 alpha, Phospho-CREB and FOXO3a expression in the same cell. These kits allow direct measurements of molecular translocationof specific proteins 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 multiplex kits contain primary antibodies toward HIF-1 alpha, phosphorylated CREB, and FOXO3a (rabbit polyclonal, mouse monoclonal, and goat polyclonal, respectively) and secondary antibodies conjugated with DyLight 549 (orange), DyLight 488 (green), and DyLight 649 (red) Fluorophores. The singleplex kits contain the same primary antibodies and DyLight 549 Conjugated Secondary Antibodies.

The Multiplex and Singleplex Transcription Factor Activation Kits have been optimized for use with the CellomicsArrayScan® HCS Reader using the Compartmental Analysis BioApplication Software Module. These kits can be used withother Cellomics BioApplications (see the Compatible BioApplication Software Modules Section). 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 HIF-1 alpha, phospho-CREB, and FOXO3a expression. In addition to HCS instruments, cells labeled by the reagents in this kit can be viewed and analyzed by other fluorescence microscopes.

In cancer cells, rapid proliferation often results in a competition for nutrients and oxygen. Three key transcription factors play a pivotal role in determining cancer cell fate: HIF, CREB and FOXO3a. All respond to both oxidative stress that occurs in rapidly proliferating cells, and to growth factor signaling through PI3 kinase (PI3K) and AKT, also known as protein kinase B (PKB). Hypoxia inducible factor 1 (HIF-1) and the cyclic AMP response element binding protein (CREB) promote cell proliferation and enhance metabolism; however, FOXO3a (FKHRL1), a member of the Forkhead family of transcription
factors, induces cell cycle arrest or apoptosis.

When oxygen levels are normal (normoxic) in the absence of growth factor signaling, HIF-1 alpha is targeted for degradationby VHL (von Hippel Lindau protein) through hydroxylation. When activated, HIF-1 (HIF-1 alpha and HIF-1 beta complex) translocates to the nucleus to activate angiogenic target genes, such as VEGF (vascular endothelial growth factor).  CREB is activated through phosphorylation by many kinases, and in conjunction with other transcription factors, upregulates many metabolic genes. Conversely, FOXO3a is negatively regulated by phosphorylation. Active FOXO3a resides in the nucleus; inactivation and transport to the cytoplasm is facilitated through phosphorylation by AKT or IKK (IK kinase). The dynamic balance between growth factor signaling and transcription factor activation regulates the fate of cancer cells.

Related Poster Presentations

	Kay Opperman, Krishna Vattem, and Richik N. Ghosh (2008) High-Content Analysis of Multiple Key Proteins in the AKT Signaling Pathway.
	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.
	Kay Opperman, Janaki Narahari, and Richik N. Ghosh (2007) Novel, Multiplexed, High Content Screening Cell-Based Assays for Activation of the Transcription Factors HIF-1a, CREB, & FOXO3a.
	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.
	Kay Opperman, Roberta Litavecz, Janaki Narahari, and Richik N. Ghosh (2007) New Transcription Factor Activation Kits for High Content Screening and Cell Based Assays.

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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