The phospho-GSK3 alpha (Ser21) kit is designed for the cell-based detection of Serine 21 phosphorylation on GSK3 alpha.
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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
The phospho-GSK3 alpha (Ser21) kit is designed for the cell-based detection of Serine 21 phosphorylation on GSK3 alpha.
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Overview

Phospho-GSK3a (Ser21) assay is designed for the robust quantification of GSK3a modulation when phosphorylated on Ser21 as a ubiquitous readout of major pathways. 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.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SPECIFICITY

Phospho-GSK3a (Ser21) assay principle

The Phospho-GSK3a (Ser21) assay measures GSK3a when phosphorylated at Ser21. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-GSK3a (Ser21) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-GSK3a (Ser21) assay principle

Phospho-GSK3a (Ser21) 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 Phospho-GSK3a (Ser21) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-GSK3a (Ser21) 2-plate assay protocol

Phospho-GSK3a (Ser21) 1-plate assay protocol

Detection of Phosphorylated GSK3a (Ser21) 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.
Phospho-GSK3a (Ser21) 1-plate assay protocol

 HTRF assay compared to Western Blot using phospho-GSK3a, total-GSK3a

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-GSK3a (Ser21) and total-GSK3a 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-GSK3a, total-GSK3a

Inhibition effect of UCN-01 on the phosphorylation of GSK3 using phospho-GSK3a assay

100,000 human MCF-7 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubation with increasing concentrations of UCN-01 (2h), cells were stimulated with insulin at 1.72µM (30min). Medium was then removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-GSK3a (Ser21) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Inhibition effect of UCN-01 on the phosphorylation of GSK3

Insulin dose-response on MCF-7 cells using phospho-GSK3a (Ser21) cellular assay

100,000 human MCF-7 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubation with increasing concentrations of Insulin (30min), medium was removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-GSK3a (Ser21) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Insulin dose-response on MCF-7 cells using phospho-GSK3a (Ser21) cellular assay

Compatibility of the HRTF phospho-GSK3a (Ser21) cellular assays with different cell lines

100,000 or 200,000 cells of different cell lines were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubation with or without UCN-01 at 1µg/ml (2h), cells were stimulated with 1.72µM of insulin (30min). Medium was removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-GSK3a (Ser21) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Compatibility of the HRTF phospho-GSK3a (Ser21) cellular assays with different cell lines

GSK3a Simplified Pathway

GSK3a 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.
GSK3a signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

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

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

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

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

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

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

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

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

Increased flexibility of phospho-assays - Application Notes

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

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

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 - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

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 - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Molecular basis of neuroinflammation and neurodegeneration diseases

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

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