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HTRF Human & Mouse Total GSK3β Detection Kit, 500 Assay Points

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.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Feature	Specification
Application	Cell Signaling
Sample Volume	16 µL

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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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Product Variants

Unit Size: 500 Assay Points

Part #:

64GTBPEG

List Price

USD 2,147.00

Unit Size: 10,000 Assay Points

Part #:

64GTBPEH

List Price

USD 12,490.00

Product information

Overview

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.

Specifications

Other Specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	Infectious Diseases Metabolism/Diabetes NASH/Fibrosis Oncology & Inflammation
Unit Size	500 Assay Points

Video gallery

How it works

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 proteins expression under a no-wash assay format.

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 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.

Assay validation

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.

Simplified pathway

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.