Data Sheet - BioVision
Contents
1. BioVision Lipase Activity Colorimetric Assay Kit Catalog K722 100 100 assays Store kit at 20 C Introduction Lipases perform essential roles in the digestion transport and processing of dietary lipids e g fats and oils in living organisms In humans pancreatic lipase is the key enzyme responsible for breaking down fats in the digestive system by converting triglyceride to monoglyceride and free fatty acid Pancreatic lipase monitoring is also used to help diagnose Crohn s disease cystic fibrosis and celiac disease Damage to the pancreas can exhibit a 5 10 fold increase of serum lipase levels within 24 to 48 hours In BioVision s Lipase Assay Kit lipase hydrolyzes a triglyceride substrate to form glycerol which is quantified enzymatically by via monitoring a linked change in the OxiRed probe absorbance A 570nm This assay is rapid simple sensitive and reliable as well as suitable for high throughput activity screening of lipase This kit detects lipase activity as low as 0 02 mU per well Kit Contents Components 100 assays Cap Code Part Number Lipase Assay Buffer 25 ml WM K722 100 1 OxiRed in DMSO 0 2 ml Red K722 100 2A Enzyme Mix lyophilized 1 vial Green K722 100 4 Lipase Substrate 0 4 ml Blue K722 100 5 Glycerol Standard 100 mM 0 2 ml Yellow K722 100 6 Lipase Positive Control lyophilized 1 vial Purple K722 100 7 Storage and Handling Store the kit at 20 C protect from ligh2. 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 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 Troublesh
3. erol 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 For research use only rev 02 13 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
4. oluble material at 13 000 x g 10 min Serum samples can be directly diluted in the Assay Buffer Prepare test samples of up to 50 ul well with Assay Buffer in a 96 well plate We suggest testing several doses of your sample to make sure readings are within the standard curve Glycerol in the sample will interfere with the result It is corrected for by using a substrate deficient control for the sample Note Some Lipases require calcium If your lipase requires calcium avoid EGTA in sample preparation and add calcium 1 5 mM to the Lipase assay buffer before use Glycerol in the sample will interfere with the result It is corrected for by using a substrate deficient control for the sample BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 02 13 For research use only 3 Reaction Mix Mix enough reagent for the number of assays to be performed For each well prepare a total 100 ul Reaction Mix Sample Control Assay Buffer 93 ul 96 ul OxiRed Probe 2 ul 2 ul Enzyme Mix 2 ul 2 ul Lipase substrate 3 ul a Add 100 ul of the Sample Reaction Mix to each well containing the Glycerol Standards Lipase positive controls and test samples Add 100 ul Control Reaction Mix to each well containing the sample controls Mix well 4 Incubate Measure OD 570 nm at T to read A measure OD 570 nm again at T2 after incubating the reaction at 37 C for 60 90 min or incubate longer time if the Lipase activity is low
5. oot 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 Page 2 of 2
6. t Allow Assay Buffer to warm to room temperature before use Briefly centrifuge vials before opening Read the entire protocol before performing the assay Reagent preparation Probe Ready to use as supplied Warm to room temperature to melt frozen DMSO before use Store at 20 C protect from light and moisture Enzyme Mix Dissolve in 220 ul Assay Buffer Partition into aliquots in vials and store at 20 C Use within two months Lipase Substrate Freezing for storage may cause the substrate to separate from the aqueous phase To redissolve the substrate keep the cap tightly closed thaw then place in a hot water bath 80 100 C for 1 minute until the substrate looks cloudy vortex for 30 seconds The substrate should be clear Repeat heat and vortex one more time The substrate is now completely in solution and ready for use Lipase positive control Dissolve the positive control in 100 ul Assay Buffer Add 5 ul and adjust the volume to 50 ul well with Assay Buffer as positive control Store at 20 C Lipase Assay Protocol Standard Curve Preparation Add 10 ul of the glycerol standard to 990 ul of Assay Buffer to generate 1 mM glycerol mix well Add 0 2 4 6 8 10 ul into a series of wells Adjust volume to 50 ul well with Assay Buffer to generate 0 2 4 6 8 10 nmol well of glycerol Standard Sample Preparations Tissues 40 mg or cells 2 x 10 can be homogenized in 4 volumes of Assay Buffer Centrifuge to remove ins
7. to read Ao protect from light 5 Calculation The OD generated by oxidation of glycerol is AA570 nm A2 A Subtracting the OD 570 nm value of control from the sample to avoid glycerol in the sample Plot Glycerol Standard Curve Apply the AA570 nm to the glycerol standard curve to get B nmol of glycerol glycerol amount generated between T and T gt in the reaction wells Glycerol generated in the test samples can then be calculated g o B x Dilution factor Lipase Activity 2 Ti xv B is the Glycerol amount from the Standard Curve in nmol T is the time of the first reading A1 in min T2 is the time of the second reading A2 in min V is the pretreated sample volume added into the reaction well in ml Unit Definition One unit is defined as the amount of lipase that hydrolyzes triglyceride to yield 1 0umol of glycerol per minute at 37 C nmol min ml mU ml Where Glycerol standard curve Sample timeline sample 01 1 N i y 0 1746x 0 0103 a fo 05 sample 02 Background 0 0 2 4 6 8 10 0 10 20 30 Glycerol nmol per well Time min RELATED PRODUCTS NAD NADH Quantification Kit ADP ATP Ratio Assay Kit Glucose Assay Kit Ethanol Assay Kit Pyruvate Assay Kit Creatine Assay Kit Triglyceride Assay Kit NADP NADPH Quantitation Kit Ascorbic Acid Quantification Kit Fatty Acid Assay Kit Uric Acid Assay Kit Lactate Assay Kit II Creatinine Assay Kit Free Glyc
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