Data Sheet - BioVision
Contents
1. 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 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 Page 2 of 22. Probe Ready to use as supplied Allow to come to room temperature before use to melt frozen DMSO Store at 20 C protect from light and moisture Use within two months PK Substrate Mix PK Enzyme Mix Dissolve with 220 ul diH O Pipette up and down to completely dissolve Store at 20 C Use within two months PK Positive Control Dissolve with 100 ul diH20 Pipette up and down to completely dissolve Store at 20 C Use within two months IV Pyruvate Kinase Assay Protocol 1 Standard Curve Preparations For the colorimetric assay Dilute the Pyruvate Standard to 1 nmol ul by adding 10 ul of the Standard to 990 ul of Assay Buffer mix well For the fluorometric assay Dilute the Pyruvate Standard to 1 nmol ul as for the colorimetric assay Then dilute the standard another 10 fold to 0 1 nmol ul by mixing 10 ul with 90 ul of Pyruvate Assay Buffer Mix well Add 0 2 4 6 8 10 ul of the diluted standard into a series of wells Adjust volume to 50 ul well with Assay Buffer to generate 0 2 4 6 8 and 10 nmol well of the Pyruvate Standard for the colorimetric assay or 0 0 2 0 4 0 6 0 8 and 1 0 nmol well for the fluorometric assay 2 Sample and Positive Control Preparations Serum can be directly added into sample wells Tissues or cells can be extracted with 4 volumes of the Assay Buffer centrifuge to get clear extract Add samples directly into 96 well plate bring volume to 50 ul well with PK Assay Buffer We suggest test
3. The signal increase is due to pyruvate generated by PK AA A2 A Note It is essential to read A and Ag in the reaction linear range It will be more accurate if you read the reaction kinetics Then choose A and Az in the reaction linear range 6 Calculation Subtract 0 standard readings from the standards Plot the pyruvate standard curve Apply the AA to the standard curve to get B nmol of pyruvate generated between T and T2 by PK in the reaction wells PK calculation B PK Activity x Sample Dilution Factor nmol min ml mU mL T2 T1 x V Where B is the pyruvate amount from pyruvate standard curve in nmol T4 is the time of the first reading A1 in min T2 is the time of the second reading A2 in min V is the sample volume added into the reaction well in ml Unit definition One unit of Pyruvate Kinase is the amount of enzyme that will transfer a phosphate group from PEP to ADP yielding 1 0 umol of pyruvate per minute at 25 C Pyruvate Standard Curve PK sample kinetics ee 1 0MU ee 0 8MU 0 6MU 0 4mU OD 570nm oe 2mU y 0 1139x 0 016 R 0 9988 oe OMU 0 5 10 0 5 10 15 20 Pyruvate nmol Time min RELATED PRODUCTS Pyruvate Assay Kit Cholesterol Assay Kit Glutathione Kit GSH GSSG and Total Maltose Assay Kit Free Fatty Acid Triglyceride Assay Kits Glycogen Starch Assay Kits Apoptosis Assay Kits many Ethanol Assay Kit Cell Proliferation Assay Kits F
4. BioVision Pyruvate Kinase Activity Colorimetric Fluorometric Assay Kit Catalog K709 100 100 assays Store kit at 20 C Introduction Pyruvate kinase PK EC 2 7 1 40 is an enzyme involved in glycolysis It catalyzes the transfer of a phosphate group from phosphoenolpyruvate PEP to ADP yielding one molecule of pyruvate and one molecule of ATP Lack of pyruvate kinase will slow down the process of glycolysis which causes the disease known as pyruvate kinase deficiency BioVision provides a simple direct and automation ready procedure for measuring pyruvate kinase activity in various biological samples such as blood tissues and culture cells etc In the assay PEP and ADP were catalyzed by PK to generate pyruvate and ATP The generated pyruvate is oxidized by pyruvate oxidase to produce color A 570 nm and fluorescence at Ex Em 535 587 nm Since the increase in color or fluorescence intensity is proportional to the increase in pyruvate amount the PK activity can be accurately measured The kit detects 0 1 mU pyruvate kinase Kit Contents Components 100 Assays Cap Code Part Number PK Assay Buffer 25 ml WM K709 100 1 OxiRed Probe 200 ul Red K709 100 2A PK Enzyme Mix Lyophilized Green K709 100 4 PK Substrate Mix Lyophilized Purple K709 100 5 PK Positive Control Lyophilized Blue K709 100 6 Pyruvate Standard 100 nmol ul 100 ul Yellow K709 100 7 II Reagent Preparation and Storage Conditions OxiRed
5. OR RESEARCH USE ONLY Not to be used on humans Lactate Assay Kit Glutamate Assay Kit Glucose Sucrose Galactose Assay Kits Ascorbic Acid Assay Kit NAD P NAD P H Assay Kit Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision GENERAL TROUBLESHOOTING GUIDE rev 10 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 Aliquot and freeze samples if needed to use multiple times Troubleshoot if nee
6. ded 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 Incorrect volumes used e Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately 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 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
7. ing several doses of your sample to ensure the readings are within the linear range For the positive control optional add 5 ul positive control solution to wells use 0 5 2 ul Positive Control for fluorometric assay adjust volume to 50 ul well with Assay Buffer 3 Reaction Mix Preparation Mix enough reagents for the number of standard and assays to be performed For each well prepare a total 50 ul Reaction Mix containing Pyruvate Kinase Measurement Background Control Assay Buffer 44 ul 46 ul Substrate Mix 7 e Enzyme Mix 2 ul 2 ul OxiRed Probe 2 ul 2 ul BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 10 13 For research use only Pyruvate in the sample will generate background If significant amount of pyruvate is in your sample the background control should be performed The background readings are then subtracted from your sample readings The fluorometric assay is 10 times more sensitive than the colorimetric assay Use 0 4 ul of the probe per reaction to decrease the background reading increase detection sensitivity significantly 4 Add 50 ul of the reaction mix to each well containing the pyruvate standard samples and controls mix well 5 Measure OD 570 nm or fluorescence Ex Em 535 587 nm at T to read A measure again at T2 after incubating the reaction at 25 C for 10 20 min or incubate longer time if the PK activity is low in sample to read Az protect from light
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