data Sheet - BioVision
Contents
1. 50 ul test samples in 96 well plate For white blood cells take 2 ml of blood and lyse RBC using RBC Lysis Buffer Cat 5831 Incubate for 10 min at room temperature Centrifuge at 400 x g for 5 min and remove the supernatant carefully Wash the pellet with 1 ml 1X PBS Centrifuge at 400 x g for 5 min and remove the supernatant carefully Lyse the pellet using 200 ul MPO Assay Buffer Keep on ice for 10 min Centrifuge at 10 000 x g for 10 min to remove insoluble material Collect the supernatant Add 1 10 ul of the WBC lysate into a 96 well plate Prepare parallel sample well s as background control Adjust the volume of background control and sample wells to 50 ul well with Assay Buffer We suggest testing several doses of a sample to ensure the readings are within the standard curve range BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 2 15 Vi 3 4 For research use only Positive Control Preparation Add 5 10 ul of the reconstituted MPO Positive Control to optional Positive Control well s Adjust the final volume 50 ul well with MPO Assay Buffer Reaction Mix Mix enough reagents for the number of assays to be performed For each well prepare a total 50 ul Reaction Mix Reaction Mix Sample Background Control Mix 40 ul MPO Assay Buffer 40 ul MPO Assay Buffer 10 ul MPO Substrate 10 ul dH 0 Add 50 ul of the Reaction Mix to each well containing the Samples amp Positive Control wells Add 52. Stop Mix in min V pre adjusted sample volume added into the reaction well in ml Unit Definition One unit of MPO is defined as the amount of MPO which generates taurine chloramine to consume 1 0 umol of TNB per minute at 25 C a 4 b c 1 B 100 Se 0 8 5 80 m 0 6 E 0 6 2 60 a 04 0 4 40 o2 A e a y 0 0151x 0 0536 0 2 K 4 Q 20 0 0 i o O 10 20 30 40 50 A or cw ate a co 5 aor we nmol TNB F actor ot va wee N9 A R er j PO gin Figure a TNB Standard Curve b Measurement of MPO activity using WBC lysate 3 ug and MPO Positive Control 5 ul and 10 ul c MPO specific activity in WBC lysate Assays were performed following kit protocol RELATED PRODUCTS NAD NADH Quantification Kit Fatty Acid Assay Kit Triglyceride Assay Kit Lipase Assay Kit Adipogenesis Assay Kit Lactate assay Kits Pyruvate Assay Kit Glycogen Assay Kit Glucose Assay Kits FOR RESEARCH USE ONLY Not to be used on humans NADP NADPH Quantification Kit Uric Acid Assay Kit Free Glycerol Assay Kit Ethanol Assay Kit Cholesterol Assay Kits Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision GENERAL TROUBLESHOOTING GUIDE rev 2 15 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 A
3. 0 ul of the Sample Background Control Mix to the sample background controls Mix well Note DO NOT ADD REACTION MIX TO STANDARDS Incubate at 25 C for 30 to 120 min record this time as T then add 2 ul Stop Mix to all sample Standard wells background control amp Positive Control wells and mix Incubate another 10 min to stop the reaction amp add 50 ul TNB Reagent Standard to each of the sample sample background control amp Positive Control wells Add 0 10 20 30 40 50 ul TNB Reagent Standard 0 10 20 30 40 50 nmol respectively to the Standard wells at this time We suggest running samples for 30 60 and 120 min followed by the Stop Mix and TNB Reagent at each time point to ensure values will fall within the linear range of the Standard Curve Measurement After 5 10 min read at 412 nm The Positive Controls and samples will show decreased color proportional to the amount of enzyme present calculated as AAgi2nm Asample background Asample It is recommended to use the AA values which are in the linear range of the Standard Curve Calculation Subtract 0 Standard reading from all Standard readings Plot the TNB Standard Curve Apply the AA412 nm of samples to the Standard Curve to get B nmol of TNB consumed in the sample reaction during the given time B Sample MPO Activity yy Sample Dilution Factor nmol min ml mU ml Where B TNB amount calculated from the Standard Curve in nmol T time of the first incubation i e pre
4. BioVision Myeloperoxidase MPO Activity Colorimetric Assay Kit Catalog K744 100 100 assays Store kit at 20 C Introduction Myeloperoxidase MPO is a peroxidase EC 1 11 1 7 abundantly expressed in neutrophils It is a lysosomal protein stored in the azurophilic granules of the neutrophil MPO contains a heme which causes its green color in secretions rich in neutrophils such as pus and some forms of mucus MPO catalyzes the production of hypochlorous acid HCIO from hydrogen peroxide H202 and chloride anion Cl or halide MPO also oxidizes tyrosine to a tyrosyl radical using hydrogen peroxide as oxidizing agent In BioVision s MPO Assay Kit HCIO produced from H202 and Cl reacts with taurine to generate taurine chloramine which subsequently reacts with the TNB probe to eliminate color A 412 nm The kit provides a rapid simple sensitive and reliable test suitable for high throughput activity assay of MPO This kit can be used to detect MPO as low as 0 05 mU per well Kit Contents Component 100 Assays Cap Code Part Number MPO Assay Buffer 25 ml WM K744 100 1 DTNB Probe 100 mM 50 ul Red K744 100 2 TCEP 50 mM 50 ul Clear K744 100 3 MPO Substrate 50 ul Blue K744 100 4 Stop Mix 20 ul Green K744 100 5 MPO Positive Control lyophilized 1 vial Purple K744 100 6 Storage and Handling Store kit at 20 C protected from light Allow Assay Buffer to warm to room temperature before use Briefly c
5. entrifuge small vials before opening Read the entire protocol prior to performing the assay Reagent Preparation TNB Reagent Standard TNB Reagent Standard TNB is easily oxidizable so it needs to be prepared from DTNB Probe as needed Use the same day as prepared discard any unused TNB reagent standard The amount of DTNB Probe for each well standard sample and background control is 0 5ul The amount per well of TCEP is 0 5 ul and of Assay Buffer is 49 ul for a total of 50 ul per well Example For 10 wells take 5 ul DTNB Probe 5 ul TCEP and 490 ul Buffer mix and set aside MPO Substrate Aliquot and store at 20 C Stable for 2 months Working solution Add 5 ul MPO Substrate to 300 ul dH2O Make fresh and discard unused portion Stop Mix Add 200 ul dH20 and dissolve Aliquot and store at 20 C Use within two months MPO Positive Control Reconstitute the positive control with 100 ul MPO Assay Buffer Aliquot and store at 20 C Use within two months MPO Assay Protocol Standard Curve Preparation Add 150 140 130 120 110 and 100 ul of MPO Assay Buffer into a series of wells The Standard will be added to the wells 0 10 20 30 40 50 ul respectively at the end of the sample incubation period see 4 below Sample Preparation Homogenize tissue or cells in 4 volumes of PBS having 0 1 NP40 centrifuge 13 000g 10 min to remove insoluble material Serum samples can be directly diluted in the MPO Assay Buffer Add 1
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7. ssay 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 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 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 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 Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures e Incorrect volumes used 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 verif
8. y 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 Air bubbles formed in well e Standard stock is at an incorrect concentration e Calculation errors Substituting reagents from older kits lots e Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes 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 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 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 bi
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