Cellomics HCS Reagent Kits for multiplex analysis of Smad3 and Phospho-CREB.

Description

The multiplexed Smad3 and Phospho-CREB HCS Reagent Kits contain optimized reagents for the simultaneous detection and quantitation of Smad3 and phospho-CREB activation in the same cell using different compounds. These kits allow direct measurements of Smad3 and phospho-CREB in 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.

Two kit versions are available in two package sizes. One version is a multiplexed kit for detecting Smad3 and phospho-CREB simultaneously. This kit contains primary antibodies against Smad3 and phospho-CREB (rabbit polyclonal and mouse monoclonal, respectively) and secondary antibodies conjugated with DyLight 488 (green) and DyLight 549 (orange) Fluorophores. The other version is a Smad3 singleplex kit that contains a rabbit polyclonal antibody against Smad3 and a DyLight 549-Conjugated Secondary Antibody. Both versions are available in a package size sufficient for one 96-well microplate and a package size sufficient for five 96-well microplates.

The Smad3 and phospho-CREB HCS Reagent Kits have been optimized for use with the ArrayScan HCS Reader and the Compartmental Analysis BioApplication Software Module. These kits can also be used with other Cellomics BioApplication Modules (see the Compatible BioApplication Software Modules Section). Thus, automated plate handling, focusing, cell image acquisition/processing, and data analysis and management have been combined into a single high content screening (HCS) system to assay for test compounds affecting the expression and activation of Smad3 and phospho-CREB transcription factors. In addition to HCS instruments, cells labeled by the reagents in this kit can be viewed and analyzed by other fluorescence microscopes.

Transforming growth factor-β (TGF-β) is part of a superfamily of polypeptides that include TGF-β isoforms, bonemorphogenetic proteins (BMPs) and growth and differentiation factors (GDFs). The cellular functions of TGF-β are extremely diverse with effects that include cellular proliferation inhibition, cell differentiation regulation, cell migration, adhesion and cell death, all attributing to the tumor suppressive role of this pathway. The signaling mechanism that controls these effects is relatively simple. TGF-β binds to its receptor complex (TβRI/ TβRII) initiating the downstream phosphorylation of the Smad tumor suppressors, specifically Smad2 and Smad3. Once phosphorylated, Smad2 and Smad3 bind to Smad4 and translocate to the nucleus where each Smad complex can regulate specific target gene expression. Additional mechanisms of TGF-β regulation occur through other signaling pathways, including protein kinase A (PKA). In this case, activated Smad3 and Smad4 bind to PKA regulatory subunits, releasing the PKA catalytic subunits to activate downstream targets of TGF-β signaling including CREB and p21Cip1, further enhancing the tumor suppressive effects of TGF-β.4 The Smad3 and phospho CREB kits along with the Cellomics ArrayScan HCS Reader and the Compartmental Analysis BioApplication Software Module enable the simultaneous quantitative monitoring of both Smad3 (directly activated by the TGF-β signaling pathway) and CREB (indirectly activated by the TGF-β signaling).

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.
	Kay Opperman, Roberta Litavecz, Janaki Narahari, and Richik N. Ghosh (2007) New Transcription Factor Activation Kits for High Content Screening and Cell Based Assays.
	Roberta Litavecz, Kay Opperman, Richik N. Ghosh (2007) Novel High Content Screening Assays for Measuring the Wnt and TGF-beta Signaling Pathways.

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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