Recombinant Human GSK-3 beta His-tag Protein, CF New

Catalog #: 11739-KS Datasheet / COA / SDS

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11739-KS-050

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Recombinant Human GSK-3 beta His-tag Enzyme Activity.

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Product Details

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human GSK-3 beta His-tag Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a peptide substrate.

The specific activity is >200 pmol/min/μg, as measured under the described conditions.

Source

Human embryonic kidney cell, HEK293-derived human GSK-3 beta protein

Met

6-His tag

Sumo-tag
(mutated, uncleavable)

3c Protease site

Human GSK-3 beta
(Ser2-Thr420)
Accession # P49841.2

N-terminal Sequence
Analysis

Protein identity confirmed by mass spectrometry

Predicted Molecular Mass

60 kDa

SDS-PAGE

55-64 kDa, under reducing conditions

Product Datasheets

11739-KS

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

11739-KS

Formulation	Supplied as a 0.2 μm filtered solution in MES, NaCl, Glycerol and DTT.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 50 mM Tris, 20 mM MgCl₂, 5 mM MnCl₂, 0.1 mg/mL BSA, pH 7.5
Recombinant Human GSK-3 beta His-tag (rhGSK-3B) (Catalog # 11739-KS)
GSK3 Substrate, 1 mg/mL stock in 25 mM Tris, pH 7.5
Adenosine triphosphate (ATP), 10 mM stock in deionized water
ADP-Glo^TMKinase Assay (Promega)
White 96-well Plate
Plate Reader with Luminescence Read Capability

Dilute rhGSK-3B to 5 µg/mL in Assay Buffer.
Prepare Substrate Mixture containing 200 µM ATP and 0.4 mg/mL GSK3 substrate in Assay Buffer.
Combine equal volumes of 5 µg/mL rhGSK-3B and Substrate Mixture. Create a Substrate Control by replacing enzyme with Assay Buffer.
Incubate at room temperature for 40 minutes in the dark.
After incubation, transfer 10 µL of each reaction to wells of a white plate.
Terminate the reaction and deplete the remaining ATP by adding 10 µL of ADP-Glo Reagent (supplied in kit) to all wells.
Incubate at room temperature for 40 minutes in the dark.
Add 20 µL Kinase Detection Reagent (supplied in kit) to all wells.
Incubate at room temperature for 30 minutes in the dark.
Read plate in Luminescence endpoint mode.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted Luminescence* (RLU) x Conversion Factor** (pmol/RLU)
	Incubation time (min) x amount of enzyme (µg)

*Adjusted for Substrate Control

**Derived from ADP-Glo^TM Kinase Assay Kit protocol (Promega)

Per reaction:

rhGSK-3B: 2.5 μg/mL
ATP: 100 µM
GSK3 Substrate: 0.2 mg/mL

Scientific Data

Enzyme Activity

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Recombinant Human GSK-3 beta His-tag (Catalog # 11739-KS) is measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a peptide substrate.

SDS-PAGE

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2 μg/lane of Recombinant Human GSK-3 beta His-tag (Catalog # 11739-KS) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 55-64 kDa, under reducing conditions.

Reconstitution Calculator

Background: GSK-3 beta

Glycogen synthase kinase-3 beta (GSK3B) is a ubiquitously expressed, highly conserved serine/threonine kinase (1-3) with key roles in the transduction of regulatory and proliferative signals at the cell membrane. GSK3B is one of two mammalian isoforms of GSK that are constitutively active and have high homology with a kinase domain and an N-terminal phosphorylation site that auto-inhibits its activity by competing with a binding site for preferred pre-phosphorylated substrates (1, 2, 4). The isoforms differ in their sequence similarity at both the N- and C-termini. The GSK3 alpha isoform is primarily cytosolic and has a glycine-rich N-terminal region that excludes it from the nucleus, whereas GSK3B can be found in the nucleus. The different localization differentiates GSK3B biological activity supported by the fact that the GSK3B knockout is embryonically lethal while GSK3 alpha expression is unable to compensate (3). GSK3 has been confirmed to have a diverse and broad range of substrates within multiple cell signaling pathways to regulate glucose metabolism, insulin activity, and energy homeostasis and plays a role in a wide range of cellular processes, such as cell proliferation, differentiation, apoptosis, the cell cycle, the immune response, and organ development (3, 5-6). GSK3B is thus implicated in multiple diseases such as cancer (2, 7) neurodegenerative diseases such as Alzheimer's, bipolar disorder (BPD), psychiatric disorders, Parkinson's, aging (8, 9), inflammatory conditions, and metabolic disorders such as diabetes, cardiovascular disease, and atherosclerosis (2-3, 7-11). There is interest in development of GSK3 inhibitors as a therapeutic target to prevent their ability to phosphorylate their substrates and prevent downstream signaling pathways; clinical trials targeting GSK3 inhibition have been primarily focused on cancers and neurodegenerative and neurological diseases (2-3, 9).

References

Dajani, R. et al. (2001) Cell 105:721.
Mancinelli, R. et al. (2017) Oxidative Med. Cell. Longev. 2017:4629495.
Wang, L. et al. (2022) Med. Res. Rev. 42:946.
Cross, D.A.E. et al. (1995) Nature 378:785.
Grimes C.A. and R.S. Jope (2001) Prog. Neurobiol. 65:391.
Doble, B.W. and J.R. Woodgett (2003) J. Cell Sci. 116:1175.
Lin, J. et al. (2020) Biochem. Biophys. Acta 1867:118659.
Gianferrara, T. et al. (2022) Curr. Med. Chem. 29:4631.
Arciniegas Ruiz, S.M.A. and H. Eldar-Finkelman (2022) Front. Mol. Neurosci 14:792364.
Sharma, A.K. et al. (2020) Drug Discov. Today 25:633.
Liang, L. et al. (2024) FASEB J. 38:e23458.