ab65345 Choline/Acetylcholine Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Choline/Acetylcholine levels in various samples. This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 11 2
1. Overview Choline and acetylcholine play important roles in many biological processes. Abcam s Choline/Acetylcholine Assay Kit provides a simple and sensitive means for quantifying Choline and Acetylcholine by either a colorimetric or fluorometric method. In the assay free choline is oxidized to betaine, via the intermediate betaine aldehyde. The reaction generates products which react with the Choline Probe to generate color (λ= 570 nm), and fluorescence (Ex/Em 535/587 nm). Acetylcholine can be converted to choline by adding acetylcholinesterase to the reaction. The kit can detect choline and acetylcholine (total choline free choline) in various biological samples such as in blood, cells, culture media, fermentation media, etc. There is no need for pre-treatment or purification of samples. The kit can detect ~10 pmol-5 nmol of choline or acetylcholine. 3
2. Protocol Summary Standard Curve Preparation Standard Curve Preparation Prepare and Add Reaction Mix Measure Optical Density or Fluorescence 4
3. Components and Storage A. Kit Components Item Quantity Choline Assay Buffer 25 ml Choline Probe (in DMSO) 200 μl Choline Enzyme Mix 1 vial Acetylcholinesterase 1 vial 5 µmol Choline Standard 1 vial * Store kit at -20 C. CHOLINE PROBE: Ready to use as supplied. Warm up to room temperature before use to melt frozen DMSO. Store at -20 C, protect from light and moisture. Use within two months. CHOLINE ENZYME MIX, ACETYLCHOLINESTERASE: Dissolve in 220 µl Choline Assay Buffer. Pipette up and down several times to dissolve. Store at -20 C. Use within two months. CHOLINE STANDARD: Dissolve in 100 µl of Choline Assay Buffer to generate 50 nmol/µl of choline standard solution. Use within two months. 5
B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. Standard Curve Preparation: a. For the colorimetric assay: Dilute the Choline Standard to 0.5 nmol/µl by diluting 10 µl of the Choline Standard into 990 µl of Choline Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 µl of the diluted standard choline into each well individually. Adjust volume to 50 µl/well with Choline Assay Buffer to generate 0, 1, 2, 3, 4, 5 nmol/well of the Choline Standard. b. For the fluorometric assay: Dilute the Choline Standard to 50 pmol/µl. Then follow the same procedure as with the colorimetric assay, add 0, 2, 4, 6, 8, 10 µl into each well individually. Adjust volume to 50 µl/well with Choline Assay Buffer to generate 0,100, 200, 300, 400, 500 pmol/well of the Choline Standard. If a more sensitive assay is desired, further dilute the standard 10-fold more, then follow the same procedure to make the standard curve at 0, 10, 20, 30, 40, 50 pmol/well. The fluorometric assay is 10-100 fold more sensitive than the colorimetric assay. 7
2. Sample Preparation: a. For serum samples: 1-10 µl/assay of Human serum can be tested (Human serum contains ~10µM choline). b. For tissue or cell samples: Tissue or cells can be lysed in Choline Assay Buffer on ice for 10 min or by homogenization, then centrifuge to remove debris. The lysate can be tested directly. Prepare test samples in 50 µl/well with Choline Assay Buffer in a 96-well plate. Note: Free choline in serum is known to increase upon storage due to breakdown of lipids. We suggest using several dilutions of your sample to ensure the readings are within the standard curve range. 3. Reaction Mix Preparation: Mix enough reagents for the number of assays performed. For each well, prepare a total 50 µl Reaction Mix containing the following components: Choline Assay Buffer 44 µl Choline Probe 2 µl Acetylcholinesterase* 2 µl Enzyme Mix 2 µl *Note: Omit the Acetylcholinesterase if you want to detect free choline only. With addition of Acetylcholinesterase, the assay detects total choline (free choline + acetylcholine). 8
4. Add 50 µl of the Reaction Mix to each well containing the Choline Standards or test samples, mix well. Incubate at room temperature for 30 min, protect from light. 5. Measure OD at 570 nm for the colorimetric assay or measure fluorescence at Ex/Em = 535/590 nm in a micro-plate reader for fluorescence assay. 5. Data Analysis Subtract background value (the zero choline control) from all standard and sample readings. Plot standard curve: nmol/well Vs. OD 570nm or fluorescence readings. Then apply the sample readings to the standard curve to obtain choline amount in the sample wells. Calculate the choline concentrations of the test samples: Choline Concentration = Cho/Sv (nmol/ml or µm) Where: Cho is the sample choline amount determined from standard curve. Sv is the sample volume (ml) added to the sample well. Acetylcholine = total choline free choline 9
Choline/Acetylcholine Assays were performed following the kit instructions. The diamonds ( ) were generated using choline as the substrate, whereas the open ( ) and closed ( ) circles were generated using acetylcholine as substrate in the presence and absence of acetylcholinesterase. 10
6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 11
Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 12
Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 13
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