Data Sheet - BioVision
Contents
1. BioVision Lipase Activity Colorimetric Assay Kit Il Catalog K723 100 100 assays Store kit at 20 C Introduction Lipase is a subclass of the esterases that catalyze the hydrolysis of ester bonds in water insoluble lipid substrates Lipases perform essential roles in the digestion transport and processing of dietary lipids e g triglycerides fats oils in most if not all living organisms In humans pancreatic lipases are the key enzyme responsible for breaking down fats in the digestive system by converting triglycerides to monoglycerides and free fatty acids During the damage of the pancreas lipase levels can rise 5 to 10 fold within 24 to 48 hours The kit provides a simple sensitive and reliable assay for rapid analysis of Lipase in samples In the assay lipases hydrolyze a specific substrate to generate a product which reacts with the DTNB probe to generate color A 412 nm The kit is also suitable for high throughput analyses Kit Contents Components 100 assays Cap Code Part Number Lipase Assay Buffer 25ml WM K723 100 1 DTNB Probe lyophilized 1 vial Red K723 100 2 Lipase Substrate 0 5 ml Blue K723 100 3 TNB Standard Lyophilized 1 vial Amber K723 100 4 Lipase Positive Control lyophilized 1 vial Purple K723 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 before opening Read the ent2. ading Az in min V is the pretreated sample volume added into the reaction well in ml Unit Definition One unit lipase is defined as the amount of lipase which hydrolyzes the substrate and generates 1 0 umol of TNB per minute at 37 C Assay Kinetics Positive Ctrl N H t a 2 Pork liver Extract Iiver Extrac y 0 0331x 0 0486 a E R 0 9988 background 0 10 20 30 40 50 0 1000 2000 3000 nmoles TNB Time sec 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 Lipase Assay Kit Adipogenesis Assay Kit NADP NADPH Quantification Kit Ascorbic Acid Quantification Kit Fatty Acid Assay Kit Uric Acid Assay Kit Lactate Assay Kit II Creatinine Assay Kit Free Glycerol Assay Kit Triglyceride Assay Kit Cholesterol Assay Kits Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 e ARCH USE ONLY Not to be used on humans BiovVisto rev 3 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 B
3. ire protocol prior to performing the assay Reagent Preparation DTNB Probe Dissolve the DTNB Probe with 1 1 ml Lipase Assay Buffer Store at 20 C Use within two months Lipase Substrate Ready to use Store at 20 C Use within two months TNB Standard Dissolve with 0 5 ml of Assay Buffer to generate 5 mM TNB Standard Stable for 2 months at 20 C Lipase Positive Control Dissolve the positive control with 100 yl Lipase Assay Buffer Store at 20 C Use within two months Lipase Assay Protocol Standard Curve Preparation Add 0 2 4 6 8 10 ul of TNB Standard into a series of wells Adjust volume to 150 ul well with Assay Buffer to generate 0 10 20 30 40 50 nmol well of TNB Standard Sample Preparations Tissues or cells can be homogenized in 4 volumes of Assay Buffer and centrifuged 13 000 x g 10 min to remove insoluble material 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 the readings are within the standard curve range Mercaptans in samples will cause a high background If the sample background is too high the sample can be precipitated with 2 volumes of saturated ammonia sulfate Then centrifuge collect the precipitates and re dissolve in the same volume of assay buffer to remove small molecule mercaptans Positive Control optional Add 5 ul of the reconstitu
4. lack 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 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 co
5. mponents 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 The most probable list of causes is under each problem section Causes Solutions may overlap with other problems GENERAL TROUBLESHOOTING GUIDE 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. ted Lipase Positive Control into Positive Control well and adjust the volume to 50 ul well with assay buffer BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 3 13 For research use only 4 Reaction Mix Mix enough reagent for the number of assays to be performed For each well prepare a total 100 pl Reaction Mix 85 ul Assay Buffer 10 pl DTNB Probe 5 ul Lipase substrate Add 100 ul of the Reaction Mix to each well containing the Positive Controls and samples Mix well DO NOT ADD TO STANDARDS Measurement Read OD 412 nm A at T after 3 min incubation time Read A2 OD 412 nm again at T after incubating the reaction at 37 C for 60 90 min or incubate longer time if the Lipase activity is low protect from light The OD of color generated upon formation of TNB is AA 412 nm A A4 It is recommended to read kinetically to choose the A and A values which are in the linear range of the Standard Curve Calculation Subtract 0 Standard from all standard readings Plot the Standard Curve Apply the AA 412 nm of samples to the standard curve to get B nmol of TNB generated in the sample reaction between T and T gt Lipase activity in samples can then be calculated Lipase Activity x Sample Dilution Factor nmol min ml mU ml B T2 T1 xV Where B is the TNB amount calculated from the Standard Curve in nmol T is the time of the first reading A1 in min Tz is the time of the second re
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