Total GSK3 alpha cellular kit HTRF®

The total GSK3 alpha kit monitors the cellular GSK3 alpha expression level and can be used as a normalization assay for the phospho-GSK3 alpha kit.
See more
  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
The total GSK3 alpha kit monitors the cellular GSK3 alpha expression level and can be used as a normalization assay for the phospho-GSK3 alpha kit.


The Total GSK3a cellular assay is used as a normalization assay with the phospho-GSK3a 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-GSK3a assay principle

The Total-GSK3a assay quantifies the expression level of GSK3a in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-GSK3a 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 GSK3a 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 GSK3a assay principle

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

Total-GSK3a 1-plate assay protocol

Detection of total GSK3a 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 GSK3a 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

GSK3 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.
GSK3 Simplified Pathway

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

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

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

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

HTRF Product Catalog

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

Neurodegeneration and its main related diseases

Discover this infographic design on neurodegenerative diseases - Infographics

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

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

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader