Data Sheet - BioVision
Contents
1. Creatinine Assay Kit Free Glycerol Assay Kit Hemin Assay Kit Total Antioxidant Capacity TAC Assay Kit Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com BioVision Rev 2 13 GENERAL TROUBLESHOOTING GUIDE For research use only Problems Cause Solution Assay not working e Use of ice cold assay buffer e Omission of a 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 e 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 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 e 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 e Use Dounce homogenizer increase the number of strokes observe for lysis under microscope e Aliquot and freeze s2. BioVision Glucose Oxidase Activity Colorimetric Fluorometric Assay Kit Catalog K788 100 100 reactions Store kit at 20 C Introduction The glucose oxidase enzyme GOx EC 1 1 3 4 is an oxidoreductase commonly found in a wide variety of microorganisms that catalyzes the oxidation of glucose to hydrogen peroxide and D glucono lactone GOx aids in breaking the sugar down into its metabolites BioVision s Glucose Oxidase Assay provides a convenient tool for sensitive detection of the GOx in a variety of samples Glucose oxidase in samples recognizes D glucose as a specific substrate leading to proportional color development The activity of GOx can be easily quantified colorimetrically A 570 nm or fluorometrically Ex Em 535 585 nm GOx assay detects glucose oxidase activity as low as 0 01mU Kit Contents Components K788 100 Cap Code Part No GOx Assay Buffer 28 ml WM K788 100 1 OxiRed Probe 0 2 ml Red K788 100 2A GOx Substrate 1 ml Blue K788 100 3 GOx Developer 1 vial Green K788 100 4 GOx Positive Control 1 vial Purple K788 100 5 H20 Standard 0 88 M 0 1 ml Yellow K788 100 6 Storage and Handling Store kit at 20 C protected from light Warm Assay Buffer to room temperature before use Briefly centrifuge all small vials prior to opening Read the entire protocol before performing the assay Reagent Reconstitution and General Consideration GOx Developer and GOx Positive Control Reco
3. amples if needed to use multiple times e Troubleshoot if needed deproteinize samples e Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples and Standards e Improperly thawed components Use of expired kit or improperly stored reagents e Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures e Incorrect 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 Pipetting errors in the standard e Pipetting errors in the reaction mix Air bubbles formed in well Standard stock is at an incorrect concentration e Calculation errors e Substituting reagents from older kits lots e 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 w
4. avelength 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 e Troubleshoot if it interferes with the kit e Refer data sheet to check if sample is compatible with the kit or optimization is needed e Concentrate Dilute sample so as to be in the linear range Note The most 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
5. de i e at the end of incubation time Calculation Subtract the 0 standard reading from all readings Plot H202 Standard Curve Calculate the glucose oxidase activity of the test sample AOD A2 A Apply the AOD to the H202 Standard Curve to get B nmol of H202 generated by Glucose Oxidase during the reaction time AT T2 T14 B Glucose Oxidase Activity ATXV x Sample Dilution Factor nmol min ml mU ml Where B is the H20 amount from Standard Curve nmol AT is the time incubated min V is the sample volume added into the reaction well ml Unit Definition One unit of GOx is the amount of enzyme that generates 1 0 umol of H202 per min at 37 C a 0 8 b 1 0 8 z 08 y 0 0591x 0 0459 g c c 0 6 o 0 4 o S 5 0 4 a o 0 2 0 2 0 0 0 2 4 6 8 10 0 5 10 15 20 25 minutes nmol H O Figure H2O2 Standard Curve a Glucose oxidase activity in sample b Assays were performed following kit protocol Related Products NAD NADH Quantification Kit ADP ATP Ratio Assay Kit Glucose Assay Kit Ethanol Assay Kit Pyruvate Assay Kit Creatine Assay Kit Ammonia Assay Kit Triglyceride Assay Kit Choline Acetylcholine Quantification Kit Sarcosine Assay Kit L amino Acid Assay Kit Nitric Oxide Assay Kit Glutamate Assay Kit FOR RESEARCH USE ONLY Not to be used on humans NADP NADPH Quantification Kit Ascorbic Acid Quantification Kit Fatty Acid Assay Kit Uric Acid Assay Kit Lactate Assay Kit II
6. he standard curve range Positive Control Add 2 10 ul of Positive Control into the desired well s amp adjust final volume to 50 ul with Assay Buffer Reaction Mix Mix enough reagents for the number of assays to be performed For each well prepare 50 ul Reaction Mix containing Reaction Mix Background Control GOx Assay Buffer 36 ul 46 ul GOx Developer 2 ul 2 ul OxiRed Probe 2 ul 2 ul GOx Substrate 10 0 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA Rev 2 13 Vi For research use only Add 50 ul of the reaction mix to each well containing samples Positive Control and standards Mix well Note The fluorometric assay is 10 fold more sensitive than the colorimetric assay so dilute the probe 10 times in Assay Buffer amp use the same volume 2 ul Background control mix is recommended for samples having high background Measurement Incubate the plate for 5 min at 37 C amp measure OD at 570 nm or fluorescence at Ex Em 535 585 nm A41 Incubate for another 15 minutes to 2 hrs at 37 C amp again measure Az Note Incubation time depends on the glucose oxidase activity in the samples We recommend measuring in a kinetic method preferably every 1 2 min and choose the period of linear range to calculate the glucose oxidase activity of the samples If the absorbance exceeds 0 7 OD 15 minutes dilute the sample and rerun the assay The H202 Standard curve can read in end point mo
7. nstitute with 220 ul Assay Buffer Pipette up and down several times to completely dissolve the pellet Don t vortex Aliquot and freeze at 20 C Stable for up to 2 months at 20 C after reconstitution or freeze thaw cycles lt 5 times Keep GOx Positive Control on ice while in use Glucose Oxidase Assay Protocol H202 Standard Curve Add 10 ul 0 88 M H20 Standard to 870 ul dH20 to make 10 mM H20 Standard Dilute 10 mM H20 Standard further to 1 19 with Assay Buffer to make 0 5 mM H20 Standard Add 0 2 4 6 8 10 ul of the diluted 0 5 mM H202 Standard into a series of wells of 96 well plate to generate 0 1 2 3 4 5 nmol well H202 Standard For the fluorometric assay dilute 0 5 mM H202 Standard 1 10 with Assay Buffer to make 50 uM H20 standard Add 0 2 4 6 8 10 ul of the diluted 50 uM H20 standard into a series of wells of 96 well plate to generate 0 0 1 0 2 0 3 0 4 0 5 nmol well H202 Standard Adjust the final volume to 50 ul with Assay Buffer Sample Preparations Homogenize cells 1x10 with 100 200 ul Assay Buffer Centrifuge at 13 000 g for 10 min to remove the insoluble material 5 50 ul serum samples can be directly diluted in the Assay Buffer Add 1 50 ul sample per well adjust final volume to 50 ul with Assay Buffer For samples having high background prepare a parallel sample well as the background control Note For unknown samples we suggest testing several doses to ensure the readings are within t
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