Cellomics HCS Reagent Kit for activated (phosphorylated) extracellular signal-regulated kinase (ERK MAPK).

Description

Mitogen-activated protein kinase (MAPK) pathways regulate a broad range of cellular processes including gene transcription, cytoskeletal organization, metabolic homeostasis, cell growth, and apoptosis. MAPKs are activated by concomitant Tyr and Thr phosphorylation, catalyzed by members of the MAP kinase kinase (MKK) family. MKKs, in turn, are activated by Ser/Thr phosphorylation, catalyzed by protein kinases of several families collectively termed Mitogen-Activated Protein Kinsase Kinase Kinases (MAPKKK). Upon activation of this pathway, ERK is phosphorylated and translocated to the nucleus of cells (Figure 2).

The Cellomics HCS Reagent Kit for ERK Activation provides high-content screening (HCS)-quality fluorescence reagents and a validated protocol for the preparation and processing of samples for the measurement of ERK kinase activation. Cells growing on standard high-density microplates are stimulated, and then fixed and immunofluorescently labeled to identify activated ERK in the cell nucleus (Figure 1). The antibody binds to dually phosphorylated ERK and has been validated for non-cross-reactivity to other MAPK (p38, JNK/SAPK) members as well as unphosphorylated ERK. Phorbol 12-Myristate 13-Acetate (PMA) is included as a control inducer of ERK translocation.

Prepared cells can be analyzed using standard fluorescence microscopy or using a Cellomics fully automated HCS Reader with the Cytoplasm to Nucleus Translocation BioApplication, which affords automated plate handling, focusing, cell image acquisition, analysis, quantification and data storage.

Features

• A more biologically relevant measure of kinase activation relative to Western blots or in vitro kinase assays
• No cell lysis, purification, or filtration steps
• Immunofluorescence based assay provides sensitive quantitative and qualitative results without radioactivity
• Kit antibody preferentially binds to dually phosphorylated (fully active) ERK, with minimized cross-reactivity to other MAPKs
• Manual sample preparation/processing requires approximately 3 hours post-compound treatment
  

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.
	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.
	Anthony Nichols, Francois Duval, Astrid Osen-Sand, Lilia Bernasconi-Castelli, Elisabeth Allet, Sandrine Pouly, Yves Sagot, Paul Lang and Alexander Scheer (2003) Use of Cellomics High Content Screening Platform to Increase Efficiency and Output for Small Molecule and Therapeutic Protein Discovery.
	Richik N. Ghosh, Vivek Abraham, Linnette Grove, Oleg Lapets and Jeffrey R. Haskins (2003) A Kinetic High Content Screening Assay for Intracellular Compartmental Analysis in Single Cells.

Kit Contents

• phospho-ERK primary antibody
• Alexa Fluor® 488 conjugated secondary antibody
• Hoechst dye
• Wash buffer (10x)
• Positive control compound (PMA)
• Permeabilization buffer (10x)
• Detergent buffer (10x)
• Plate seals
  

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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