MMP-13 Colorimetric Assay Kit

GENMED SCIENTIFICS INC. U.S.A

Glycogen Synthase Kinase-3Activity Assay Kit

BACKGROUND

Glycogen Synthase Kinase-3 (GSK-3；EC2.7.11.26), 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.

COMPONENT (50tests)

PLEASE READENTIRE BOOKLET BEFORE PROCEEDING WITH THE ASSAY. CAREFULLY NOTE THEHANDLING AND STORAGE CONDITIONS OF EACH KIT COMPONENT.

ASSAY BUFFER

FORM: Liquid

STORAGE: -20℃

QUANTITY: 5 ml

ENZYME MIX

FORM: Liquid

STORAGE: -20℃

QUANTITY: 1 ml

REACTION BUFFER

FORM: Liquid

STORAGE: -20℃

QUANTITY: 1 ml

SUBSTRATE

FORM: Liquid

STORAGE: -20℃

QUANTITY: 1 ml

GSK3αINHIBITOR

FORM: Liquid

STORAGE: -20℃

QUANTITY: 250 μl

GSK3βINHIBITOR

FORM: Liquid

STORAGE: -20℃

QUANTITY: 250μl

DILUTION SOLUTION

FORM: Liquid

STORAGE: 4℃

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

EXPERIMENTAL METHODS

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

5．Add 10µlSubstrate

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.

DATA ANALYSIS

Units/ml enzyme =【（ΔA）（0.1）（Df）】 /【(6.22)（0.005）（5）】

ΔA=A(test)—A（blank）

0.1= Volume（inmilliliters）of the assay

Df=Dilution factor

6.22=Millimolar extinction coefficient of oxidized NADH at 340nm

0.005=Volume (in milliliters) of sample used

5 = Total reaction time (minutes)

Unit Definition

One unit will oxidize 1.0 μM ofNADH toNAD per minite at pH 7.5 at 30℃.

TECHNICAL NOTE

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

REFERENCES

Adams JA, McGlone ML, Gibson R, Taylor SS. 1995. Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase. Biochemistry34: 2447–2454

Brady, MJ. et al. 1998.The activation of glycogen synthase by insulin switches from kinase inhibition to phosphatase activation during adipogenesis in 3T3-L1 cells.J. Biol. Chem. 273, 14063-6

Cook P.F, Neville M.E Jr., Vrana K.E, Hartl F.T, Roskoski R Jr.1982. Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit. Biochemistry 21:5794–5799.

Doble BW. and Woodgett JR. 2003. GSK-3: tricks of the trade for a multi-tasking kinase. Journal of Cell Science, 116:1175-1186.

Javorek D., Welsch J. 1985. Adenosine 5’-diphosphate and adenosine 5’-monophosphate: Uvmethod, in:H.U.Bergmeyer(Ed).,Methods of enzymatic Analysis. Third edition. Vol. VII. Verlag Chemie. pp 365-370

Kirschenbaum, F., et al. 2001. Substitution of a glycogen synthase kinase-3beta phosphorylation site in presenilin 1 separates presenilin function from beta-catenin signaling. J. Biol. Chem.276, 7366-75.

Martinez A., Alonso M., Castro A., Perez C., and Moreno F. 2002. First non-ATP cpmpetitive glycogen kinase 3 (GSK3) inhibitors: Thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease. J. Med. Chem. 45,1292-1299

McKay GA., Thompson PR., and Wright GD. 1994. Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity.Biochemistry 33,6936-6944.

Meijer L., Flajolet M., and Greengard P. 2004. Pharmacological inhibitors of glycogen synthase kinase 3. Trends in Pharmacological Sciences. 25, 471-480

Mettey Y., Gompel M., Thomas V., Garnier M., Leost M., Ceballos-PicotI., Noble M., Endocott J., Vierfond JM. And Meijer L. 2003. Aloisines, a new family of CDK/GSK-3 inhibitors. SAR study, crystal structure in complex with CDK2, enzyme selectivity, and cellular effects. J. Med. Chem. 46,222-236

Naerum L.,Norskov-Lauritsen L., and Olesen PH. 2002. Scaffold hopping and optimization towards libraries of glycogen synthase kinase-3 inhibitors. Bioorg. Med. Chem. Lett. 12, 1525-1528

Roskoski R Jr . 1983. Assays of protein kinase. Methods Enzymol99, 3–6.

Schulte AE., van der Heijden R., Verpoorte R.1999. Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension culture cells. Anal. Biochem. 269,245-254

Sichelschmidt OJ., Hahnefeld C., Hohlfeld T., Herberg FW. And Schror K. 2003. Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity.Cardiovascular Research 58(3):602-610

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.