Phosphatidylcholine Assay Kit
Contents
1. Product Manual Phosphatidylcholine Assay Kit Catalog Number STA 600 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Researc Creating Solutions for Life Science Research Introduction Phospholipids are important structural lipids that are the major component of cell membranes and lipid bilayers Phospholipids contain a hydrophilic head and a hydrophobic tail which give the molecules their unique characteristics Most phospholipids contain one diglyceride a phosphate group and one choline group Phosphatidylcholine is the foremost membrane phospholipid in eukaryotic cell membranes and is present in every cell in the body It serves as a pool for many lipid messengers and is a source for bioactive lipids such as phosphatidic acid diacylglycerol lysophosphatidylcholine and others Regulation of phosphatidylcholine biosynthesis and metabolism is important to membrane structure maintenance and function Phosphatidylcholine is the chief source of choline in the body which itself and its derivative compounds form cell signaling molecules such as acetylcholine platelet activating factor and sphingophosphorylcholine As one of the most prevalent phospholipids in lipoproteins phosphatidylcholine exerts considerable influence on lipid homeostasis Since phosphatidylcholine comprises about 70 of the total phospholipids in plasma lipoproteins it is very important2. Lipid Res 8 676 681 Recent Product Citation Park E S et al 2014 Phosphatidylcholine alteration identified using MALDI imaging MS in HBV infected mouse livers and virus mediated regeneration defects PLoS One 9 e103955 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2012 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing eo CELL BIOLABS INC
3. 02 One 25 mL bottle 3 Fluorescence Probe 100X Part No 260003 One 100 uL tube in DMSO 4 5 Standard Diluent 10X Part No 260006 One 1 mL tube 96 well Microtiter Plate Part No 234501 One 96 well clear bottom black plate HRP Part No 234402 One 100 uL tube of 100 U mL HRP solution in glycerol Box 2 shipped on blue ice packs 1 2 3 Phosphatidylcholine Standard Part No 260001 One 25 uL tube Phospholipase D 350X Part No 260004 One 30 uL tube Choline Oxidase Part No 260005 One 50 uL tube Materials Not Supplied 1 P Mmi p S Distilled or deionized water 1X PBS 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Multichannel micropipette reservoir Fluorescence microplate reader capable of reading excitation in the 530 570 nm range and emission in the 590 600 nm range optional Chloroform optional Superoxide dismutase Storage Upon receipt store the Phosphatidylcholine Standard Fluorescence Probe HRP and Choline Oxidase at 20 C The Fluorescence Probe is light sensitive and must be stored accordingly Avoid multiple freeze thaw cycles Store the Phospholipase D at 80 C Store the remaining kit components at 4 C Preparation of Reagents e 1X Assay Buffer Warm the Assay Buffer 10X to room temperature prior to using Dilute the Assay Buffer 10X
4. citrate and centrifuge at 1000 x g and 4 C for 10 minutes Remove the plasma layer and store on ice Take care to avoid disturbing the white buffy layer Aliquot samples for testing and store remaining solution at 80 C Perform plasma dilutions in 1X Assay Buffer Plasma samples must be diluted at least 1 50 to 1 400 with Assay Buffer This will provide values within the range of the standard curve Notes 1 Samples with NADH concentrations above 10 uM and glutathione concentrations above 50 uM will oxidize the probe and could result in erroneous readings To minimize this interference it is recommended that superoxide dismutase SOD be added to the reaction at a final concentration of 40 U mL CELL BIOLABS INC 2 Avoid samples containing DTT or B mercaptoethanol since the fluorescence probe is not stable in the presence of thiols above 10 uM 3 Choline can generate high background if present in samples If choline may be present run a background control without Phospholipase D Subtract this value from sample reading values Preparation of Phosphatidylcholine Standard Curve 1 Prepare fresh phosphatidylcholine standards by first diluting a portion of the 100 mg mL Phosphatidylcholine Standard stock solution 1 100 in 1X Standard Diluent eg Add 5 uL of Phosphatidylcholine Standard in 495 uL Standard Diluent Vortex thoroughly This provides a 100 mg dL concentration Use this 100 mg dL solution to prepare a series of the rema
5. ining phosphatidylcholine standards according to Table 1 below 100 mg dL Resulting Phosphatidylcholine 1X Assay Buffer Phosphatidylcholine Tubes Standard uL uL Concentration mg dL 1 50 450 10 2 250 of Tube 1 250 5 3 250 of Tube 2 250 2 5 4 250 of Tube 3 250 1 25 5 250 of Tube 4 250 0 625 6 250 of Tube 5 250 0 313 7 250 of Tube 6 250 0 156 8 0 500 0 Table 1 Preparation of Phosphatidylcholine Standards Note Do not store diluted phosphatidylcholine standard solutions Assay Protocol Each phosphatidylcholine standard and sample should be assayed in duplicate or triplicate A freshly prepared standard curve should be used each time the assay is performed 1 Add 10 uL of the diluted phosphatidylcholine standards or samples to the 96 well microtiter plate 2 Add 100 uL of the prepared Reaction Reagent to each well and mix the well contents thoroughly 3 4 Read the plate with a fluorescence microplate reader equipped for excitation in the 530 570 nm Cover the plate wells to protect the reaction from light Incubate the plate for 60 minutes at 37 C range and for emission in the 590 600 nm range Calculate the concentration of phosphatidylcholine within samples by comparing the sample RFU to the phosphatidylcholine standard curve CELL BIOLABS INC er o gt Example of Results The following figures demonstrate typical Phosphatidylcholine Assay results O
6. line is then oxidized by choline oxidase to produce hydrogen peroxide The hydrogen peroxide is then detected with a highly specific fluorescence probe Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide which bind in a 1 1 ratio Samples are compared to a known concentration of phosphatidylcholine standard within the 96 well microtiter plate format Samples and standards are incubated for 60 minutes and then read with a standard 96 well fluorometric plate reader Figure 1 CELL BIOLABS INC ae te NO OOO OM oper 0 do Phosphatidylcholine Phosphatidylcholine Specific Phospholipase D PARA ma OH A AZAAAA AA 0 la AON Hona H3C Da CH Phosphatidic Acid Choline Choline Oxidase Hoo pi 3 a alae H O Betaine Aldehyde HRP H O Figure 1 Phosphatidylcholine Assay Principle Related Products 1 STA 361 Human ApoAI and ApoB Duplex ELISA Kit STA 368 Human ApoB 100 ELISA Kit STA 369 OxiSelect Human Oxidized LDL ELISA Kit MDA LDL Quantitation STA 384 Total Cholesterol Assay Kit Colorimetric STA 391 HDL and LDL VLDL Cholesterol Assay Kit STA 394 HDL Cholesterol Assay Kit STA 396 Serum Triglyceride Quantification Kit Colorimetric STA 398 Free Glycerol Assay Kit Colorimetric STA 601 Sphingomyelin Assay Kit O o o A OA p o a S _ CELL BIOLABS INC A Sa Kit Components Box 1 shipped at room temperature 1 2 Assay Buffer 10X Part No 2600
7. ne should use the data below for reference only This data should not be used to interpret or calculate actual sample results 3000 2500 2000 1500 RFU 1000 500 0 lt l l l 0 2 4 6 8 10 12 Phosphatidylcholine mg dL Figure 2 Phosphatidylcholine Standard Curve Calculation of Results 1 Calculate the average fluorescence values for every standard control and sample Subtract the average zero standard value from itself and all standard and sample values This is the corrected fluorescence 2 Plot the corrected fluorescence for the standards against the final concentration of the phosphatidylcholine standards from Table 1 to determine the best curve See Figure 2 for an example standard curve 3 Determine the phosphatidylcholine concentration of the samples with the equation obtained from the linear regression analysis of the standard curve Substitute the corrected fluorescence values for each sample Remember to account for dilution factors Sample corrected fluorescence Total Phosphatidylcholine mg dL ee SSS x Sample dilution Slope gt gt CELL BIOLABS INC P S References 1 Chapman D et al 1967 Chem Phys Lipids 1 445 475 2 Hojjati M R et al 2006 J Lipid Res 47 3 673 676 3 Linsel Nitchke P et al 2005 Nature Reviews Drug Discovery 4 193 205 4 Ohta Fukuyama M 1980 J Biochem 88 197 203 5 Phillips G B et al 1967 J
8. or samples to achieve optimal assay results and minimize possible interfering compounds Run proper controls as necessary Always run a standard curve with samples Tissues or Cell Suspensions Homogenize 250 mg of sample wet tissue or cell pellet in 4 5 mL of chloroform methanol 2 1 v v Centrifuge to remove debris After centrifugation incubate the homogenate at room temperature for 1 hour on an orbital shaker Induce phase separation by adding 1 25 mL dH O Incubate 10 minutes at room temperature and centrifuge at 1000 x g for 10 minutes Collect the lower chloroform organic phase and re extract the upper phase with 2 mL of solvent mixture whose composition is CHCl3 MeOH water 86 14 1 v v v Combine organic phases and dry in a vacuum centrifuge Dissolve in 200 uL CHCl3 MeOH water 60 30 4 5 v v v for storage Before phosphatidylcholine assay samples must be diluted at least 1 50 to 1 400 with Assay Buffer Serum Collect blood without using an anticoagulant Allow blood to clot for 30 minutes at room temperature Centrifuge at 2000 x g and 4 C for 10 minutes Remove the serum layer and store on ice Take care to avoid disturbing the white buffy layer Aliquot samples for testing and store remaining solution at 80 C Perform serum dilutions in 1X Assay Buffer Serum samples must be diluted at least 1 50 to 1 400 with Assay Buffer This will provide values within the range of the standard curve Plasma Collect blood with heparin or
9. to lipid homeostasis Determining circulatory levels of phospholipids and lipoproteins is critical to the diagnosis of lipid transport disorders Fatty acid residues from phosphatidylcholine are transferred via the enzyme lecithin cholesterol acyltransferase LCAT to cholesterol to create cholesterol esters High levels of cholesterol and cholesterol esters hypercholesterolemia have been associated with cardiovascular disease such as atherosclerosis and heart disease In addition phosphatidylcholine synthesis is altered in many neurological cardiovascular and pulmonary disease states Cell Biolabs Phosphatidylcholine Assay Kit is a simple fluorometric assay that measures the amount of phosphatidylcholine present in plasma or serum tissue homogenates or cell suspensions in a 96 well microtiter plate format Each kit provides sufficient reagents to perform up to 96 assays including blanks phosphatidylcholine standards and unknown samples Sample phosphatidylcholine concentrations are determined by comparison with a known phosphatidylcholine standard Assay Principle Cell Biolabs Phosphatidylcholine Assay Kit measures the phosphatidylcholine present within serum plasma or tissue samples The assay is based on the enzyme driven reaction that will detect phosphatidylcholine via phosphatidylcholine specific phospholipase D enzyme and choline oxidase First phospholipase D hydrolyzes phosphatidylcholine into choline and phosphatidic acid Cho
10. with deionized water by diluting the 25 mL Buffer with 225 mL deionized water for 250 mL total Mix to homogeneity Store the 1X Assay Buffer at 4 C up to six months e 1X Standard Diluent Warm the Standard Diluent 10X to room temperature prior to using Dilute the Standard Diluent 10X with deionized water by diluting the 1 mL Diluent with 9 mL deionized CELL BIOLABS INC o gt ae water for 10 mL total Vortex to homogeneity Store the 1X Standard Diluent at 4 C up to six months Reaction Reagent Prepare a Reaction Reagent by diluting the Choline Oxidase 1 200 HRP 1 500 Fluorescence Probe 1 100 and Phospholipase D 1 350 in 1X Assay Buffer e g for 50 assays combine 25 uL of Choline Oxidase 10 uL of HRP 50 uL Fluorescence Probe and 15 uL Phospholipase D with 1X Assay Buffer to 5 mL total solution Mix thoroughly and protect the solution from light For best results place the Reaction Reagent on ice and use within 30 minutes of preparation Do not store the Reaction Reagent solution Note The Fluorescence Probe is light sensitive and must be stored accordingly Preparation of Samples Samples should be assayed immediately or stored at 80 C prior to performing the assay Optimal experimental conditions for samples must be determined by the investigator The following recommendations are only guidelines and may be altered to optimize or complement the user s experimental design A set of serial dilutions is recommended f
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