Datasheet - BioVision
Contents
1. A Ao apply the AOD to the NADH standard curve to get B nmol of NADH generated by GDH during the reaction time AT T2 T4 B GDH Activity AT xV Where B is the glutamate amount from standard curve in nmol T is the time incubated V is the sample volume added into the reaction well in ml Unit Definition One unit is the amount of enzyme that will generate 1 0 umol of NADH per min at pH 8 at 37 C x Sample Dilution Factor nmol min ml mU ml 0 8 NADH Standard 0 8 Sample Test ample E 0 6 E o6 kel B 04 0 4 c 3 fo 02 y 0 0699x 0 099 0 2 ti 2 R 0 9999 Background o 0 control 0 500 1000 1500 0 2 4 6 8 10 Time sec NADH nmol Sample Bovine Liver extraction 36g protein RELATED PRODUCTS NAD NADH Quantification Kit Glucose Assay Kit Ethanol Assay Kit Lactate Assay Kit L amino Acid Assay Kit Sarcosine Assay Kit Creatinine Assay Kit Uric Acid Assay Kit NADP NADPH Quantification Kit ADP ATP Ratio Assay Kit Pyruvate Assay Kit Lactate Assay Kit II Glutamate Kit Glycogen Assay Kit Creatine Assay Kit Fatty Acid Assay Kit FOR RESEARCH USE ONLY Not to be used on humans Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision GENERAL TROUBLESHOOTING GUIDE rev 11 14 For research use only Problems Cause Solution Assay not working e Use of ice cold assay buffer e Omission of a2. 9 ml of ddH2O Pipette up and down several times to completely dissolve the pellet into solution Do not vortex Reconstitute the GDH Positive Control with 220 yl Assay Buffer Keep on ice during the preparation and protect from light Aliquot and store 20 C Reconstitute the NADH with 50 pl ddH2O to generate a 10 mM NADH stock solution The GDH Positive Control and the Developer are stable for up to 2 months at 20 C after reconstitution or freeze thaw cycles lt 5 times Reconstituted NADH 10 mM and the supplied Glucose 2 M solution are stable for up to 6 months at 20 C Glucose Dehydrogenase Assay Protocol NADH Standard Curve Dilute 10 ul of the 10 mM NADH stock solution with 90 ul of Assay Buffer to generate a 1 mM NADH standard Add 0 2 4 6 8 10 ul of the 1 mM NADH standard into a 96 well plate in duplicate to generate 0 2 4 6 8 10 nmol well standards Adjust the final volume to 50 ul with Assay buffer Sample Preparations Tissues 50 mg or cells 1 x 10 can be homogenized in 200 ul ice cold Assay Buffer then centrifuged 13 000 x g 10 min to remove insoluble material 5 50 ul serum samples can be directly diluted in the Assay Buffer Adjust the final volume of test samples to 50 ul well with Assay Buffer in a 96 well plate We suggest testing several doses of your sample to make sure the readings are within the linear range of the standard curve and set up the background control group to avoid inter
3. BioVision Glucose Dehydrogenase Activity Colorimetric Assay Kit Catalog K786 100 100 reactions Store kit at 20 C Introduction Glucose 1 dehydrogenase NAD EC 1 1 1 118 is an enzyme that catalyzes the chemical reaction D glucose NAD lt D glucono 1 5 lactone NADH H This enzyme belongs to the family of oxidoreductases specifically those acting on the CH OH group of donor with NAD or NADP as acceptor BioVision s Glucose Dehydrogenase GDH Assay Kit provides a convenient tool for sensitive detection of the GDH in a variety of samples The GDH present in sample will recognize D glucose as a specific substrate leading to a proportional color development The activity of GDH can be easily quantified colorimetrically A 450 nm This assay detects GDH activity as low as 0 01 mU with our unit definition Kit Contents Components K786 100 Cap Code Part No GDH Assay Buffer 25 ml WM K786 100 1 Glucose 2 M 1ml Blue K786 100 2 Developer lyophilized 1 vial Red K786 100 3 GDH Positive Control lyophilized 1 vial Green K786 100 4 NADH Standard 0 5 pmol Lyophilized 1 vial Yellow K786 100 5 Storage and Handling Store the kit at 20 C protect from light Allow Assay Buffer to warm to room temperature before use Briefly centrifuge vials prior to opening Read the entire protocol before performing the assay Reagent Reconstitution and General Consideration Reconstitute Developer with 0
4. ference of the NADH in the sample For the positive control optional add 2 ul positive control solution to wells and adjust to a final volume of 50 ul with Assay Buffer BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 11 14 For research use only 3 Reaction Mix Mix enough reagents for the number of assays to be performed For each well prepare a Reaction Mix 100 ul containing Reaction Mix Background Control Mix 92 ul Assay Buffer 8 ul GDH Developer 82 ul Assay Buffer 8 ul GDH Developer 10 pl 2 M Glucose Add 100 ul of the Reaction Mix to each well containing the test samples positive controls and standards add 100 ul of the Background Control Mix to each well containing the background control sample Mix well 4 Measurement Incubate the mix for 3 min at 37 C then measure OD at 450 nm ina microplate reader Ao incubate for another 30 mins to 2 hrs at 37 C and measure OD at 450 nm again Ai Note Incubation times depends on the GDH activity in your samples We recommend measuring the OD in a kinetic method preferably every 3 5 min and choose the period of linear range to calculate the GDH activity of the samples The NADH Standard Curve can read in Endpoint Mode i e at the end of the incubation time 5 Calculation Subtract the 0 Standard value from all readings standards and test samples Plot the NADH standard Curve then calculate the GDH activity of the test samples AOD
5. orrect volumes used e Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately e Always thaw and prepare fresh reaction mix before use e Refer datasheet amp verify correct incubation times and temperatures e Use calibrated pipettes and aliquot correctly Readings do not follow a linear pattern for Standard curve e Use of partially thawed components e Pipetting errors in the standard e Pipetting errors in the reaction mix e Air bubbles formed in well e Standard stock is at an incorrect concentration e Calculation errors e Substituting reagents from older kits lots Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes e Prepare a master reaction mix whenever possible e Pipette gently against the wall of the tubes e Always refer the dilutions in the data sheet e Recheck calculations after referring the data sheet e Use fresh components from the same kit Unanticipated results e Measured at incorrect wavelength Samples contain interfering substances e Use of incompatible sample type Sample readings above below the linear range e Check the equipment and the filter setting Troubleshoot if it interferes with the kit e Refer data sheet to check if sample is compatible with the kit or optimization is needed Concentrate Dilute sample so as to be in the linear range Note The m
6. ost probable list of causes is under each problem section Causes Solutions may overlap with other problems BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 2
7. step in the protocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Assay buffer must be at room temperature e Refer and follow the data sheet precisely e Check the wavelength in the data sheet and the filter settings of the instrument Fluorescence Black plates clear bottoms Luminescence White plates Colorimeters Clear plates Samples with erratic readings e Use of an incompatible sample type Samples prepared in a different buffer Samples were not deproteinized if indicated in datasheet Cell tissue samples were not completely homogenized e Samples used after multiple free thaw cycles e Presence of interfering substance in the sample e Use of old or inappropriately stored samples e Refer data sheet for details about incompatible samples Use the assay buffer provided in the kit or refer data sheet for instructions e Use the 10 kDa spin cut off filter or PCA precipitation as indicated Use Dounce homogenizer increase the number of strokes observe for lysis under microscope Aliquot and freeze samples if needed to use multiple times e Troubleshoot if needed e Use fresh samples or store at correct temperatures until use Lower Higher readings in Samples and Standards e Improperly thawed components e Use of expired kit or improperly stored reagents e Allowing the reagents to sit for extended times on ice Incorrect incubation times or temperatures e Inc
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