GENMED SCIENTIFICS INC. U.S.A
Glycogen Synthase Kinase-3Activity Assay Kit
Glycogen Synthase Kinase-3 (GSK-3；EC126.96.36.199), is a highly conserved, ubiquitously expressed serine/threonine protein kinase.GSK-3's homolog in the fruit fly Drosophila melanogaster is known as Shaggy (Zeste White 3). GSK-3 works in the Wnt signalling pathway to phosphorylate β-Catenin. Phosphorylation leads to ubiquitination and degradation by cellular proteases, preventing it from entering the nucleus and activating transcription factors. GSK-3 can be inhibited by AKT phosphorylation, which is part of the insulin signal transduction. The phosphorylation of the target proteins by GSK-3 usually inhibits their activity,such as glycogen synthase andnuclear factor of activated T lymphocytes.GSK-3is involved in cell differentiation, cellular growth and proliferation, apoptosis control, inflammation, and neuronal function. In mammals GSK-3 is encoded by two known genes GSK-3αand 3β, which share high homology at their catalytic site. GSK-3αhas an additional 60 amino acid residues N-terminal to the kinase domain which is glycine rich. Higher levels of GSK-3 have been shown in pre-tangle and in phosphorylated tau-bearing neurons. Overexpression of GSK-3 is a characteristic feature of Alzheimer's disease.The development of GSK-3 inhibitors holds considerable promise for reducing tau phosphorylation and debilitating effects of Alzheimer's disease.
The Glycogen Synthase Kinase-3 Activity Assay Kitis a complete assay system designed to measure the activity of GSK-3, GSK-3αand GSK-3βby coupling the formation of ADP to the reaction catalyzed by PK and LDH in the presence of phosphoenolpyruvate (PEP) with the oxidation of NADH. The disappearance of NADH is detected by measuring a decrease in extinction at 340 nm (ε=6,220 M-1cm-1).The unpriming peptide substrates used in the assay are RRAAEELDSRAGSPQL,derived from elF2B and GPHRSTPESRAAV,derived from presenilin 1. Inhibitor senstitive enzymatic activity assay is used to determine the activity of both GSK-3αand GSK-3βisoforms, respectively.
PLEASE READENTIRE BOOKLET BEFORE PROCEEDING WITH THE ASSAY. CAREFULLY NOTE THEHANDLING AND STORAGE CONDITIONS OF EACH KIT COMPONENT.
QUANTITY: 5 ml
QUANTITY: 1 ml
QUANTITY: 1 ml
QUANTITY: 1 ml
QUANTITY: 250 μl
QUANTITY: 1 ml
OTHER MATERIALS REQUIRED
Pipetman or multi-channel pipetman capable of pipetting 10-100 µl accurately (note: reagents can be diluted to increase the minimal pipetting volume to >10 µl).
Ice bucket to keep reagents cold until use.
Water bath or incubator for component temperature equilibration.
Spectrophotorimetry for optical density read
Note on storage: Store all components at -20°C for the highest stability. When setting up the assay, do not maintain diluted components at reaction temperature (e.g. 30°C) for an extended period of time prior to running the assay.
To start assay:
1．Briefly warm kit components to RT.
2．Pipet65 µl AssayBufferintoa separate acuvette.
3．Add 10 µlEnzyme Mix
4．Add 10 µlReaction Buffer
6．(optional) Add 5 µl either GSK3αInhibitor or GSK3βInhibitor
7．Allow cuvettes to equilibrate to assay temperature (30°C) for 3 min
8．Start reaction by the addition of 5µl sample （50μg protein）and Dilution Solutionto thetest and blank cuvettes.
9．Continuously read plates at A340nmin a spectrophotorimetry. Record data at 1 min. time intervals for 5 min.
10．Perform data analysis.
Units/ml enzyme =【（ΔA） （0.1）（Df）】 /【(6.22)（0.005）（5）】
0.1= Volume（inmilliliters）of the assay
6.22=Millimolar extinction coefficient of oxidized NADH at 340nm
0.005=Volume (in milliliters) of sample used
5 = Total reaction time (minutes)
One unit will oxidize 1.0 μM ofNADH toNAD per minite at pH 7.5 at 30℃.
- The kit can be performed in 96-well plate format
- Wear gloves, goggles and lab coat. Avoid contact and inhalation.
- All samples must be treated using state-of-the-art technology
- All samples must be clearly labeled
- Suitable instruments must be used for taking samples and for their preparation
- Follow the manufacture’s instructions for application/use
- Use either single inhibitor or both inhibitors to determine the enzymatic activity of both isofomes
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USE OF PRODUCT
This product contains research chemicals. As such, they should be used and handled only by or under the supervision of technically qualified individuals. This product is not intended for
diagnostic or human use.
LIMITED PRODUCT WARRANTY
This warranty limits our liability to replacement of this product. No other warranties of any kind. Express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by GenMed Scientifics. GenMed Scientifics shall have no liability for any direct, indirect,consequential,or incidental damages arising out of the use, the results of use, or the inability to use this product.