Phospho-GSK3β (Ser9), Phospho-GSK3α (Ser21), Total GSK3β & Total GSK3α Cellular Assay Kits

Phospho- and total GSK3β and GSK3α detection as ubiquitous readout of major pathways

Cisbio's cell-based homogeneous HTRF® kits for phospho-GSK3 beta (Ser9), alpha (Ser21) and both total GSK3 assays enable separate detection of both isoforms of GSK3 and a multiple pathway readout. GSK3 is involved in PI3K/AKT signaling, Wnt/β-Catenin, Hedgehog, Notch, glycogen synthesis and cytoskeleton polymerization which control protein synthesis, cell proliferation, migration, inflammation, immune response, glucose regulation and apoptosis. The assays provide the quantitative detection of GSK3, the phosphorylated isoforms beta (Ser9) and alpha (Ser21), and the total proteins for normalization of the phospho-level detected. Phosphorylation of GSK3β Ser9 and GSK3α Ser21 normally decreases its enzymatic activity. The assays are suitable for cell-based screening for GSK3-upstream kinase inhibitors that result in increased GSK3 activity during cancer treatment, or GSK3 inhibitors for CNS disorders. The simple add-and-read protocol eliminates all wash steps for a faster analysis and easy miniaturization, while maintaining a high-quality specific output and offering enhanced convenience over ELISA or WB.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total GSK3 assays are based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-GSK3 antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with GSK3, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-GSK3 can be quantitatively detected using the HTRF phospho-GSK3alpha (Ser21), phospho-GSK3beta (Ser9) and total GSK3 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

What to expect at the bench This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

GSK3 is active in numerous central intracellular signaling pathways, PI3K/AKT signaling, Wnt/β-catenin, hedgehog, glycogen synthesis and cytoskeleton polymerization, regulating important cellular processes like cellular proliferation, migration, inflammation, immune responses, glucose regulation, and apoptosis. GSK3 kinase phosphorylates its protein substrates and thereby usually inhibits the activity of downstream targets. GSK3 also plays essential roles by regulating various transcription factors such as c-Jun, AP-1, β-Catenin, CREB, and NF-κB. Due to its significant functions across major pathways, GSK3 activity is subject to tight regulation. This activity is generally high in non-proliferating cells, whereas GSK3 is inhibited in response to growth factors, cytokines and hormones by the phosphorylation of GSK3α on Ser21 and GSK3β on Ser9. Phosphorylation of GSK3β onTyr216 or of GSK3α Tyr279 enhances its enzymatic activity. 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 GSK3α on Ser21 and GSK3β 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 for storage. In the Wnt pathway and in the absence of Wnt signaling, GSK3 also plays a significant role in controlling β-Catenin degradation by its phosphorylation, together with the destruction complex. Upon Wnt signaling, GSK3 is inhibited through Dvl protein, leading to the cytoplasmic accumulation of β-Catenin and resulting in translocation of β-Catenin into the nucleus, to interact with the transcriptional factors TCF/LEF to promote gene expression. Furthermore, activation of GSK3 induces the inhibition of the Hedgehog signaling pathway, whereas inhibition of GSK3 activates Tau function in microtubule polymerization. Therefore GSK3 is considered a high potential target for the treatment of nervous system disorders, such as bipolar disorder, Alzheimer's or Parkinson’s disease, but also type II diabetes, inflammation, bone disease and some types of cancer.

Product Performance

1. HTRF assay compared to Western Blot using phospho-GSK3α, total-GSK3α , phospho-GSK3β and total-GSK3β 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-GSK3α (Ser21), total-GSK3α, phospho-GSK3β (Ser9) and total-GSK3β 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.

2. Inhibition effect of UCN-01 on the phosphorylation of GSK3 using phospho-GSK3α and phospho-GSK3β assays

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-GSK3α (Ser21) and phospho-GSK3β (Ser9) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

3. Insulin dose-response on MCF-7 cells using phospho-GSK3α (Ser21) and phospho-GSK3β (Ser9) cellular assays

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-GSK3α (Ser21) and phospho-GSK3β (Ser9) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

4. Compatibility of the HRTF phospho-GSK3α (Ser21) and the HTRF phospho-GSK3β (Ser9) 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-GSK3α (Ser21) and phospho-GSK3β (Ser9) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
GSK3 alpha phospho-S21 kit - 500 tests64GPAPEG
GSK3 alpha phospho-S21 kit - 10,000 tests64GPAPEH
GSK3 alpha phospho-S21 kit - 50,000 tests64GPAPEY
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GSK3 beta phospho-S9 kit - 500 tests64GPBPEG
GSK3 beta phospho-S9 kit - 10,000 tests64GPBPEH
GSK3 beta phospho-S9 kit - 50,000 tests64GPBPEY
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GSK3 alpha total kit - 500 tests64GTAPEG
GSK3 alpha total kit - 10,000 tests64GTAPEH
GSK3 alpha total kit - 50,000 tests64GTAPEY
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GSK3 beta total kit - 500 tests64GTBPEG
GSK3 beta total kit - 10,000 tests64GTBPEH
GSK3 beta total kit - 50,000 tests64GTBPEY
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Companion products

DescriptionCat. noProduct insertMSDS
GSK3 alpha phospho-S21 kit control lysate64GPATDA
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GSK3 beta phospho-S9 kit control lysate64GPBTDA
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GSK3 alpha total kit control lysate64GTATDA
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GSK3 beta total kit control lysate64GTBTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF

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