Cellomics HCS Reagent Kit for detection of phosphorylated alpha and beta isoforms of glycogen synthase kinase-3.

primarily nuclear localization of phospho-GSK-3	treat cells	detect redistribution of phospho-GSK-3	compare to nuclear & cell boundaries	analyze and conclude

Description

The Cellomics Phospho-GSK-3 Detection Kit measures phosphorylation of the a(Ser21) and b(Ser9) isoforms of glycogen synthase kinase-3 (GSK-3), a kinase involved in glycogen metabolism, translation regulation and Wnt signaling. The kit contains a polyclonal rabbit phospho-GSK-3 antibody, a goat anti-rabbit secondary antibody conjugated to DyLight 649 Fluorophore, Whole Cell Stain Green and various other reagents and buffers required for immunofluorescent detection of phospho-GSK-3 for high-content screening (HCS) assays.

Glycogen synthase kinase-3 (GSK-3) participates in many key cellular pathways regulating proliferation and differentiation and was originally identified for its phosphorylation of glycogen synthase, the rate-limiting enzyme of cellular glycogen storage. GSK-3 is normally active in resting cells; however, after growth factor, metabolic or differentiation signals, GSK-3 is activated through phosphorylation by AKT (PKB), PKA, PKC or the p70 and p90 S6 kinases. In the active state, GSK-3 phosphorylates targets that have already been “primed” by a phosphorylation event, resulting in inactivation of that protein, including b-catenin, eIF2B and glycogen synthase. Because of its regulatory role in these critical pathways determining cell fate, GSK-3 is a target of interest for type II diabetes, inflammation, Alzheimer’s disease and cancer.

Phospho-GSK-3 phosphorylation was measured using IGF-1-treated 3T3 L1 pre-adipocytes, and the assay was optimized with the Thermo Scientific ArrayScan HCS Reader and Morphology Explorer BioApplication Software Module. In this application, many features, including texture measurements and intensity are available for quantitation. Both Whole Cell Stain Green and Hoechst 33342 stain are included to enable quantitation using other bioapplications, such as Compartmental Analysis and Spot Detector. After stimulation with IGF-1, GSK-3 is phosphorylated, resulting in its inactivation and movement to the membrane periphery and other subcellular locations in the cytoplasm (Figure 1). GSK-3 phosphorylation can be measured using total cellular fluorescent intensity or changes in intracellular texture (including intracellular spots). Cells stained using this kit also can be imaged using fluorescence or confocal microscopy.

Figure 1. Phospho-GSK-3 detection in 3T3 L1 pre-adipocytes. After 24 hours of growth factor depletion, cells were treated for 30 minutes with IGF-1 in media lacking glucose. Cells were fixed and stained according to the kit protocol. In non-treated cells (top panels), GSK-3 is localized in the nucleus. Stimulation results in the phosphorylation and inactivation of GSK-3 and increased staining in the cytoplasm.

Figure 2. Time course and dose response curves of phospho-GSK-3 in 3T3 L1 cells treated with IGF-1. Left panel: Time course of cells treated with IGF-1 (100 ng/ml). Each point represents data from 32 wells. The numbers above each data point indicate Z´ factor. Right panel: Triplicate dose response curve cells stimulated for 30 minutes. Each point represents the average from eight wells of three replicate plates.

Related Poster Presentations

Kay Opperman, Krishna Vattem, and Richik N. Ghosh (2008) High-Content Analysis of Multiple Key Proteins in the AKT Signaling Pathway.

Kit Contents

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.

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