Total GSK3 beta cellular kit HTRF®

The Total GSK3 beta kit monitors the cellular GSK3 beta expression level and can be used as a normalization assay for the phospho-GSK3 beta kit.
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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
The Total GSK3 beta kit monitors the cellular GSK3 beta expression level and can be used as a normalization assay for the phospho-GSK3 beta kit.
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The Total GSK3ß cellular assay is used as a normalization assay with the phospho-GSK3ß kit. It is optimal for metabolic research. GSK3 is involved in PI3K/AKT signaling, Wnt/ß-Catenin, Hedgehog, Notch, glycogen synthesis, and cytoskeleton polymerization, which control protein synthesis, cell proliferation, migration, inflammation, immune response, glucose regulation, and apoptosis.



Total-GSK3ß assay principle

The Total-GSK3ß assay quantifies the expression level of GSK3ß in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-GSK3ß assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of GSK3ß in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total GSK3ß assay principle

Total-GSK3ß 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total-GSK3ß HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total GSK3ß 2-plate assay protocol

Total-GSK3ß 1-plate assay protocol

Detection of total GSK3ß with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Total GSK3ß 1-plate assay protocol

HTRF assay compared to Western Blot using phospho-GSK3ß and total-GSK3ß cellular assays

Human MCF-7 cells were grown in a T175 flask at 37 °C, 5% CO2 until 80% confluency. After a 1.72 µM insulin stimulation for 30 min, cell culture medium was discarded and cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF. By using HTRF phospho-GSK3ß (Ser9) and total-GSK3ß cellular assays, just 1,500 cells are sufficient for minimal signal detection, while 12,500 cells are needed for a Western Blot signal. The HTRF cellular assays are at least 4-fold more sensitive than the Western Blot.
HTRF assay compared to Western Blot using phospho-GSK3ß and total-GSK3ß cellular assays

GSK3 Simplified Pathway

GSK3 is active in numerous central intracellular signaling pathways and regulates important cellular processes from proliferation and immune responses, to glucose regulation and apoptosis. GSK3 kinase phosphorylation inhibits the activity of downstream targets. GSK3 is also regulated by cellular localization and by protein complex formation. In the insulin pathway, insulin activates PI3K, which phosphorylates AKT. In turn, activated AKT phosphorylates GSK3a on Ser21 and GSK3ß on Ser9, resulting in GSK-3 inactivation and inducing activation of glycogen synthase by dephosphorylation. Dephosphorylated GSK3 inactivates glycogen synthase, which converts glucose to glycogen. Thus, GSK3 is considered a versatile potential target for various disease research applications.
GSK3 signaling pathway

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Présentations scientifiaues

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

New cell-based HTRF® assays for the exploration of Wnt signaling pathway

Investigation of the endogenous AKT/GSK3 and Wnt pathways - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Notes d'application

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Notes techniques

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Notes techniques

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Notes techniques

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Vidéos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalogue

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Vidéos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

Molecular basis of neuroinflammation and neurodegeneration diseases

The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides

Neurodegeneration and its main related diseases

Discover this infographic design on neurodegenerative diseases - Infographies

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques

Safety Data Sheet GSK3 b total Kit / 64GTBPEG

64GTBPEG - Fiches de sécurité

Safety Data Sheet GSK3 b total Kit / 64GTBPEH

64GTBPEH - Fiches de sécurité

Plate Reader Requirement

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