Data Sheet - BioVision
Contents
1. BioVision Alcohol Dehydrogenase Activity Colorimetric Assay Kit Catalog K787 100 100 reactions Store kit at 20 C Introduction Alcohol dehydrogenase Alcohol DH ADH EC 1 1 1 1 is a group of seven dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD to NADH In humans and many other animals they serve to break down alcohols which could otherwise be toxic in yeast and many bacteria some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation BioVision s Alcohol Dehydrogenase Assay Kit provides a convenient tool for sensitive detection of the ADH in a variety of samples In the assay ADH will utilize isopropanol as a substrate leading to a proportional color development The activity of ADH can be easily quantified colorimetrically A 450 nm This assay detects ADH activity as low as 0 01 mU in samples Kit Contents Components K787 100 Cap Code Part No ADH Assay Buffer 25 ml WM K787 100 1 Substrate 1ml Blue K787 100 2 Developer Lyophilized 1 vial Red K787 100 3 ADH Positive Control Lyophilized 1 vial Green K787 100 4 NADH Standard 0 5 umol Lyophilized 1 vial Yellow K787 100 5 Storage and Handling Store the kit at 20 C protect from light Allow ADH Assay Buffer to warm to room temperature before use Briefly centrifuge vials prior to opening Read the entire2. IDE rev 08 13 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 e Samples prepared in a different buffer 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 e Use the assay buffer provided in the kit or refer data sheet for instructions e Use Dounce homogenizer increase the number of strokes observe for lysis under microscope e 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 e Incorrect incubation times or temperatures e
3. 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 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 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 wavelength e Samples contain interfering substances e Use of incompatible sample type e 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 Th
4. e 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 Page 2 of 2
5. protocol before performing the assay Reagent Reconstitution and General Consideration Reconstitute Developer with 0 9 ml of ddH20 Pipette up and down several times to completely dissolve the pellet into solution Do not vortex Reconstitute the ADH Positive Control with 220 ul Assay Buffer Keep on ice during the preparation and protect from light Aliquot and store 20 C Reconstitute the NADH with 50 ul ddH20 to generate a 10 mM NADH stock solution The ADH Positive Control and the Developer are stable for up to 2 months at 20 C after reconstitution or freeze thaw cycles lt 5 times Substrate amp reconstituted NADH 10 mM are stable for up to 6 months at 20 C Alcohol Dehydrogenase Assay Protocol NADH Standard Curve Dilute 10 ul of the 10 mM NADH stock solution with 90 pl of ADH Assay Buffer to generate 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 200ul ice cold Assay Buffer then centrifuged 13 000 x g 10 min to remove insoluble material Add 2 50 ul samples into 96 well plate For the positive control optional dilute Positive Control 1 9 by adding 2 ul of Positive Control to 18 ul Assay Buffer Add 2 10 ul of diluted positive control solution to desired well s For se
6. rum sample 5 50 wl serum can be directly tested Adjust the final volume of test samples to 50 ul well with Assay Buffer in the 96 well plate Notes A We suggest testing several doses of your sample to make sure the readings are within the linear range of the standard curve BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 08 13 For research use only B NAD P H or other enzymes in samples may give non specific readings set up the background control see next step below to subtract the non specific background interference in samples 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 82 ul ADH Assay Buffer 8 ul Developer 10 pl Substrate 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 samples Mix well Background Control Mix 92 ul ADH Assay Buffer 8 ul Developer Measurement Incubate the mix for 3 min at 37 C then measure OD at 450 nm in a microplate reader Ao incubate for another 30 mins to 2 hrs at 37 C and measure OD at 450 nm again A incubation times will depend on the ADH activity in the samples We recommend measuring the OD in a kinetic method preferably every 3 5 min and choose the period of linear range within the s
7. tandard curve to calculate the ADH activity of the samples The NADH Standard Curve can read in Endpoint Mode i e at the end of the incubation time Calculation Subtract the 0 Standard value from all readings standards and test samples Plot NADH standard Curve Calculate the OD increase by the test samples AOD A4 Ao apply the AOD to the NADH standard curve to get B nmol of NADH generated by ADH during the reaction time AT T2 T4 ADH Activity x Sample Dilution Factor nmol min ml mU ml AT xV Where B is the NADH amount generated by ADH in nmol T is the time of reaction in minute 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 0 8 pene 0 8 Sample Test Sample 0 6 F Eoi 3 o wo 04 Fri 3 a e o 0 2 y ao EDA 3 o Background 0 T o control 9 2 4 6 8 10 0 500 1000 1500 NADH nmol Time sec 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 NADP NADPH Quantification Kit ADP ATP Ratio Assay Kit Pyruvate Assay Kit Lactate Assay Kit II Glutamate 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 GU
Download Pdf Manuals
Related Search