Acetylcholine Assay Kit (Colorimetric)
Contents
1. Product Manual Acetylcholine Assay Kit Colorimetric Catalog Number STA 603 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research S l Introduction Acetylcholine is a polyatomic cation neurotransmitter that is produced in acetylcholinergic neurons It is one of many neurotransmitters within the autonomic nervous system and the only neurotransmitter in the motor function of the somatic nervous system Acetylcholine works within the peripheral and central nervous systems within many organisms including humans It is manufactured via choline acetyltransferase from acetyl CoA and choline Choline is an amine that is an essential nutrient that is a key precursor to many phospholipids Acetylcholine works in the peripheral nervous system by activating skeletal muscles as well as smooth muscle and cardiac muscle function Within the central nervous system acetylcholine acts as a neuromodulator for the cholinergic system which causes excitatory actions Here the neurotransmitter is involved with plasticity excitability arousal and reward Acetylcholine s half life and activity are very short because it is broken down by acetylcholinesterase There are two main acetylcholine receptors nicotinic and muscarinic Acetylcholine disorders can have a profound impact on neurological function A shortage of acetylcholine such as the autoimmune disorder Myasthen2. absorbance 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 absorbance 2 Plot the corrected absorbance for the standards against the final concentration of the acetylcholine standards from Table 1 to determine the best curve See Figure 2 for an example standard curve 7 N CELL BIOLABS INC EN 3 Determine the acetylcholine concentration of the samples with the equation obtained from the linear regression analysis of the standard curve Substitute the corrected absorbance values for each sample Remember to account for dilution factors Acetylcholine uM Sample corrected absorbance x Sample dilution Slope Note 1 mM acetylcholine 14 62 mg dL or 146 ppm References 1 Gilberstadt M L et al 1984 Anal Biochem 138 78 85 2 Holm P L et al 2003 Clin Chem 49 286 294 3 Kovarik Z et al 2003 Biochem J 373 33 40 4 Magnottl R A et al 1987 Clin Chem 33 10 1731 1735 5 Vizi E S et al 1985 J Pharmacol Methods 13 201 211 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 sol
3. centrifuge Dissolve in 200 uL CHCl4 MeOH water 60 30 4 5 v v v for storage Before acetylcholine 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 Prior to testing filter samples with a 3K 10K centrifugal filter e g Millipore Amicon Ultra 0 5mL Ultracel membrane filters or Thermo Pierce Concentrators PES membrane filters Perform serum dilutions in 1X Assay Buffer Serum samples must be diluted at least 1 20 with Assay Buffer for accurate determinations Plasma Collect blood with heparin or 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 Prior to testing filter samples with a 3K 10K centrifugal filter e g Millipore Amicon Ultra 0 5mL Ultracel membrane filters or Thermo Pierce Concentrators PES membrane filters Perform plasma dilutions in 1X Assay Buffer Plasma samples must be diluted at least 1 100 to 1 200 with Assay Buffer for accurate determinations Notes 1 Samples with NADH concentrations above 10 uM and glutathion
4. e 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 2 Avoid samples containing DTT or f mercaptoethanol since the 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 Acetylcholinesterase Subtract this value from sample reading values 5 CELL BIOLABS INC Preparation of Acetylcholine Standard Curve 1 Prepare fresh acetylcholine standards by first diluting a portion of the 10 mM Acetylcholine Standard stock solution 1 50 in 1X Assay Buffer eg Add 10 uL of Acetylcholine Standard stock in 490 uL 1X Assay Buffer Vortex thoroughly This provides a 200 uM concentration Use this 200 uM solution to prepare a series of the remaining acetylcholine standards according to Table 1 below Acetylcholine 1X Assay Buffer Resulting Acetylcholine Tubes Standard uL uL Concentration uM 1 10 490 200 2 250 of Tube 1 250 100 3 250 of Tube 2 250 50 4 250 of Tube 3 250 25 5 250 of Tube 4 250 12 5 6 250 of Tube 5 250 6 25 7 250 of Tube 6 250 3 13 8 250 of Tube 7 250 1 57 9 250 of Tube 8 250 0 78 10 0 500 0 Table 1 Preparation of Acetylcholine Standards Note Do not
5. e 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 02013 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing e CELL BIOLABS INC
6. ia gravis leads to muscle fatigue and weakness due to antibodies blocking acetylcholine receptors Acetylcholine has also been implicated in many disease states including diabetic vasculopathy hypertension and Alzheimer s disease Cell Biolabs Acetylcholine Assay Kit is a simple colorimetric assay that measures the amount of acetylcholine 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 acetylcholine standards and samples Sample acetylcholine concentrations are determined by comparison with a known acetylcholine standard The kit s detection sensitivity limit is 0 75 uM acetylcholine Assay Principle Cell Biolabs Acetylcholine Assay Kit is based on the enzyme driven reaction that will detect acetylcholine via acetylcholinesterase enzyme and choline oxidase First acetylcholinesterase hydrolyzes acetylcholine into choline and acetic acid Choline is then oxidized by choline oxidase to produce hydrogen peroxide The hydrogen peroxide is then detected with a highly specific colorimetric 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 acetylcholine standard within a 96 well microtiter plate format Samples and standards are incubated for 60 minutes and then read with a standard 96 wel
7. imetric Probe and 10 uL Acetylcholinesterase with 1X Assay Buffer to 2 5 mL total CELL BIOLABS INC e WEN solution Mix thoroughly and protect the solution from light For best results place the Acetylcholine Reaction Reagent on ice and use within 30 minutes of preparation Do not store the Acetylcholine Reaction Reagent solution 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 for 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 dH2O 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 CHCl MeOH water 86 14 1 v v v Combine organic phases and dry in a vacuum
8. l colorimetric plate reader in the 540 570 nm range Figure 1 CELL BIOLABS INC E CH J Y HC o cH CH Acetylcholine Acetylcholinesterase O HG on JL P wt H3C oO H3C cH Acetic Acid Choline Choline Oxidase CH Q 2 y H Colorimetric Hs Probe H O Betaine Aldehyde HRP OD 540 570 nm H O Figure 1 Colorimetric Acetylcholine 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 390 Total Cholesterol Assay Kit Fluorometric STA 391 HDL and LDL VLDL Cholesterol Assay Kit STA 396 Serum Triglyceride Quantification Kit Colorimetric STA 600 Phosphatidylcholine Assay Kit STA 601 Sphingomyelin Assay Kit 10 STA 602 Acetylcholine Assay Kit Fluorometric O o0 NCD Mm PY Pp 3 CELL BIOLABS INC P Kit Components Box 1 shipped at room temperature 1 Assay Buffer 10X Part No 260202 One 50 mL bottle 2 Colorimetric Probe 50X Part No 260301 One 100 uL tube in DMSO 3 HRP Part No 234402 One 100 uL tube of 100 U mL HRP solution in glycerol Box 2 shipped on blue ice packs 1 Acetylcholine Standard Part No 260201 One 50 uL tube 2 Acetylcholinesterase Part No 260204 One 10 Unit tube 3 Choline Oxidase Part No 260205 One 25 uL tube Materials Not Supplied 1 96 well
9. microtiter plates Distilled or deionized water 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 Spectrophotometric microplate reader capable of reading in the 540 570 nm absorbance range E o EN ie Centrifugal filters for plasma or serum samples e g Millipore Amicon Ultra 0 5mL Ultracel membrane filters or Thermo Pierce Concentrators PES membrane filters 8 Reagents and equipment necessary for sample preparation 9 optional Chloroform 10 optional Methanol 11 optional Superoxide dismutase Storage Upon receipt store the Assay Buffer 10X at 4 C Store the remaining components at 20 C The Colorimetric Probe is light sensitive and must be stored accordingly Avoid multiple freeze thaw cycles Preparation of Reagents e 1X Assay Buffer Warm the Assay Buffer 10X to room temperature prior to using Dilute the Assay Buffer 10X with deionized water by diluting the 50 mL Buffer with 450 mL deionized water for 500 mL total Mix to homogeneity Store the 1X Assay Buffer at 4 C up to six months e Acetylcholine Reaction Reagent Prepare a reaction reagent to test for acetylcholine by diluting the Choline Oxidase 1 200 HRP 1 500 Colorimetric Probe 1 50 and Acetylcholinesterase 1 250 in 1X Assay Buffer eg For 50 assays combine 12 5 uL of Choline Oxidase 5 uL of HRP 50 uL Color
10. store diluted acetylcholine standard solutions Acetylcholine Assay Protocol Each acetylcholine standard and sample should be assayed in duplicate or triplicate A freshly prepared standard curve should be used each time the assay is performed l 2 Add 50 uL of the diluted acetylcholine standards or samples to a 96 well microtiter plate Add 50 uL of the prepared Acetylcholine Reaction Reagent to each standard and sample wells Mix all well contents thoroughly Cover the plate wells to protect the reaction from light Incubate the plate on an orbital rotator for 60 minutes at room temperature 4 Read the plate with a spectrophotometric microplate reader in the 540 570 nm range Calculate the concentration of acetylcholine within samples by comparing the sample absorbance to the acetylcholine standard curve Example of Results The following figures demonstrate typical Acetylcholine Assay results One should use the data below for reference only This data should not be used to interpret or calculate actual sample results CELL BIOLABS INC o gt er 1 6 1 4 1 2 o fo OD 540 nm o o o A o iv o 100 150 Acetylcholine uM 200 250 0 18 0 16 0 14 0 12 c 0 1 i 0 08 a O 0 06 0 04 0 02 6 8 Acetylcholine uM 10 Figure 2 Acetylcholine Standard Curve Calculation of Results Calculate the average
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