GPDH Activity Assay - B-Bridge International, Inc.
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1. The GPDH Activity Assay is a rapid and easy to use assay to quantify GPDH in cultured cells or tissue samples When exogenous DHAP and NADH are mixed with the test sample glycerol 3 phosphate and NAD will be produced if the sample contains GPDH activity GPDH activity is measured by the decrease concentration of NADH B Bridge International Inc 3 V121213 www b bridge com GPDH Activity Assay Components Kit components can be stored at 20 C prior to use e Substrate Solution containing DHAP and NADH lyophilized eeeeeee 10 bottles e Enzyme Extraction Reagent DOWE L ccceeeeee ee ee neces ee eee ee ee ee eeeee ea eeeeeed 1 bag 1 kit 100 tests Additional materials and equipment may be required Distilled water Pipette PBS Spectrophotometer with 340 nm wavelength Quartz microcuvette Centrifuge Centrifuge tubes Sonicator Protocol l Reagent preparation and storage Prepare and store reagents at 4 C 1 Reconstitute the lyophilized Substrate Solution in 4 2 ml purified water per bottle The solution is stable for 2 days at 4 C Only reconstitute the number of bottles that will be used immediately 2 Dissolve the Enzyme Extraction Reagent in 200 ml distilled water The solution is stable for 4 weeks at 4 C Note Do not mix reagents from different kits unless they have the same lot number ll Sample Preparation Samples should be prepared and maintained at 4 C 1 Tissue sampl2. 4 Add 800 ul Substrate Solution per well 24 well or 80 ul per well 96 well Bring the solution to room temperature Allow samples to equilibrate to room temperature Add 400 ul of sample per well 24 well or 40 ul per well 96 well Gently mix do not create bubbles Use the kinetic analysis mode of the spectrophotometer or manually measure OD3a9nm starting at time 0 and every 30 seconds for 3 minutes IV Calculation of GPDH activity 1 2 3 Plot OD34onm against time Using the linear range of the graph select 2 time points that are 1 minute apart i e time points 60 and 120 seconds to calculate AOD3agnm min AODsaonmmin xA 6 22xBxC GPDH activity U ml A ml Total reaction volume 6 22 Millimolar absorption coefficient of NADH molecules B ml Sample volume assayed C cm Optical path length If followed dilutions in Section Ill A 600 ml B 200 ml C 1cm If using a microplate then Sample volume in the well ml Bottom surface area of well cm 1 Unit of GPDH activity is defined as 1 ml of sample consumes 1 umole of NADH in 1 minute light path 1cm B Bridge International Inc 5 V121213 www b bridge com GPDH Activity Assay Application Notes for cultured adipocytes Primary culture from adipose tissue or cell lines i e 3T3 L1 3T3 f442 0b1771 Culture medium 1 Basal Medium DMEM containing high concentration of glucose 4 50 g l high glucose with 10 FBS 2 Differenti
3. IES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES 2007 B Bridge International Inc All Rights Reserved B Bridge International Inc 2 V121213 www b bridge com GPDH Activity Assay Purpose GPDH Activity Assay is a quantitative colorimetric measurement of glycerol 3 phosphate dehydrogenase GPDH activity in cell cultures and tissue samples such as adipocytes and adipose tissues Introduction An organism s major sources of fatty acids come from its diet or mobilization from cellular storage Fatty acids from the diet are solubilized and absorbed through the gut and delivered to cells via blood transport Excess free state long chain fatty acids are cytotoxic in cells Adipocytes avoid the accumulation of fatty acids by storing it in the form of triacylglycerols In adipose tissue GPDH reduces dihydroxyactone phosphate DHAP to glycerol 3 phosphate using coenzyme NAD The sequential binding of three glycerol 3 phosphates by coenzyme acyl CoA generates triacylglycerol In response to energy demands the fatty acids stored as triacylglycerols can be utilized by peripheral tissues NADH NAD Dihydroxyacetone phosphate ee Glycerol 3 phosphate 2 09 GPDH The measurement of GPDH activity is often used as a marker for lipogenesis biosynthesis of fat The activity of GPDH rapidly increases upon differentiation of precursor adipocytes to mature adipocytes
4. ation Medium Basal medium 0 25 uM Dexamethazone and 10 ug ml Insulin Culture protocol 1 Plate cells at 0 5 1 X 10 cells well in a 24 well plate It takes approximately 1 2 days for cells to become confluent Once culture is confluent replace the Basal Medium with the Differentiation Medium Incubate for 2 days Replace the medium with Basal Medium Add test compounds such as inhibitors and inducers for lipid accumulation Incubate 5 10 days until lipid accumulates in the cells Wash cells 2 times using PBS Add 0 5 1 ml Enzyme Extraction Solution to each well Remove cells with a rubber policeman and place the cells in a tube 10 Sonicate cells on ice 11 Perform GPDH assay on the extract OMANOAR WN Example Data Decrease in OD with each GPDH Activity compound added 0 08 0 07 lt 0 06 g A d P E 0 05 x 2 0 04 l 5 Q 0 03 O mge Blank Z 0 02 fat 0 01 0 O 30 60 90 120 150 180 Time sec A B C V References 1 Tashiro K Inamura M Kawabata K Sakurai F Yamanishi K Hayakawa T Mizuguchi H Efficient Adipocyte and Osteoblast Differentiation from Mouse Induced Pluripotent Stem Cells by Adenoviral Transduction Stem Cells 27 1802 1811 2009 2 Nagane K Jo J Tabata Y Promoted Adipogenesis of Rat Mesenchymal Stem Cells by Transfection of Small Interfering RNA Complexed with a Cationized Dextran Tissue Eng Part A 16 21 31 2010 B Bridge International Inc 6 V121213 ww
5. e i Homogenize 1 g of adipose tissue in 4 ml of 0 25M sucrose ii Centrifuged at 700 x g for 10 minutes at 4 C iii If a fat layer forms on the surface of the sample carefully remove the fat layer v Transfer the supernatant cytosol fraction to a tube and dilute 20 to 100 times with the Enzyme Extraction Reagent then assay samples 2 Culture cells i Remove culture medium and wash the cells 2 times with 500ul PBS ii Add Enzyme Extraction Reagent to each well For a 24 well plate use 0 5 1 ml per well iii Scrape cells with a sterile rubber policeman Transfer cells to a clean centrifuge tube B Bridge International Inc 4 V121213 www b bridge com GPDH Activity Assay iv Using a sonicator homogenize the cell extracts v Crude extracts may be directly assayed or centrifuged at 12 800 x g for 5 minutes at 4 C Centrifugation is recommended vi Assay the sample supernatant lll Assay Procedure 1 TOTO Add 400 ul of resuspended Substrate Solution to a quartz microcuvette Bring the solution to room temperature If the spectrophotometer has an incubator incubate 5 minutes at 25 C Allow samples to equilibrate to room temperature Add 200 ul of sample to the cuvette containing the Substrate Solution Mix well Use the kinetic analysis mode of the spectrophotometer or manually measure OD340nm starting at time 0 and every 30 seconds for 3 minutes Alternative 24 well and 96 well plate format 1 2 3
6. w b bridge com GPDH Activity Assay 3 Matsumura K Bae JY Hyon SH Polyampholytes as Cryoprotective Agents for Mammalian Cell Cryopreservation Cell Transplant 19 691 699 2010 4 Jiao WH Gao H Li CY Zhou GX Kitanaka S Ohmurae A Yao XS beta Carboline Alkaloids from The Stems of Picrasma quassioides Magn Reson Chem 48 490 495 2010 5 Kozak L P and Jensen J T 1974 J Biol Chem 249 7775 7781 6 Wise L and Green H 1979 J Biol Chem 254 273 275 For assistance and ordering please contact B Bridge International Inc 20813 Stevens Creek Blvd Suite 200 Cupertino CA 95014 USA Tel 408 252 6200 Fax 408 252 6220 Email customersupport b bridge com www b bridge com B Bridge International Inc V121213 www b bridge com
7. wA B Bridge International Inc GPDH Activity Assay Measure glycerol 3 phosphate dehydrogenase in precursor adipocytes Catalog PMC AK01 COS For research use only Not for diagnostic use V121213 GPDH Activity Assay Table of Contents PUTDO SO 5 5 tance ode ctenctilais a a e ar e ae a arsine ate sseueee O R a 3 WATFODUCTION EA EE EEE E Saesavea cog onaeahaen A fais pand EE AE AE sadee eaves 3 COMPONENTS A vase cso cad aA a AE decennial celui AARE AE Ea dein seine wavered eee ees 4 Additional MatefidlS cence hte A a A ico cloval aia case ineeedsaby 4 POlOCOlt sce222 6 eile 28 322 Fo eek see ee tas ei te NN Be Sh PEASE NC A EA O TS ad oe 4 Application NOLES xs snc ge eu viie keener Pee yee sue auch ok eas pues e a 6 FREICLENCES 2oiontecottecetoeraccerneoonsaassdsbecatacanetosesensdaniulusmanece I AAAA AENA EN TEE AS 6 Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESS OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANT
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