ab83377 Phosphatidylcholine Assay Kit
Instructions for Use For the rapid, sensitive and accurate measurement of Phosphatidylcholine 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
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2.
Protocol Summary
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3.
Components and Storage
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4.
Assay Protocol
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5.
Data Analysis
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6.
Troubleshooting
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1. Overview Phosphatidylcholine (PC) is a phospholipid which incorporates choline as the headgroup of the lipid. PC is a major constituent of biological membranes and is involved in cell signaling through release of choline by phospholipase D leaving the second messenger phosphatidic acid. Abcam's Phosphatidylcholine Assay Kit is a simple convenient means of measuring Phosphatidylcholine in a variety of biological samples. The kit utilizes an enzyme-coupled assay in which PC is hydrolyzed, releasing choline which is subsequently oxidized resulting in development of the OxiRed probe to generate fluorescence (Ex/Em = 535/587 nm) and absorbance (570 nm). Abcam’s Phosphatidylcholine kit measures PC in the range of 0.1 to 10 nmol per sample. PC is present in serum at ~0.2-2.5 mM (~50-200 mg/dL).
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2. Protocol Summary Sample Preparation
Standard Curve Preparation Prepare and Add Reaction Mix
Measure Optical Density or Fluorescence
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3. Components and Storage A. Kit Components Item
Quantity
PC Assay Buffer
25 mL
OxiRed Probe
0.2 mL
PC Hydrolysis Enzyme (Lyophilized)
1 vial
PC Development Mix (Lyophilized)
1 vial
PC Standard (10 µmol, (Lyophilized)
1 vial
* Store kit at -20°C, protect from light. •
Allow Assay Buffer to warm to room temperature before use.
•
Briefly centrifuge all small vials prior to opening.
•
Read the entire protocol before the assay.
PC PROBE: Ready to use as supplied. Warm to >18°C to melt frozen DMSO prior to use. Store at -20°C; protect from light and moisture. Stable for 2 months.
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PC HYDROLYSIS ENZYME, DEVELOPMENT MIX: Dissolve with 220 µl PC Assay Buffer separately. Pipette up and down to dissolve. Keep the Enzyme and Development Mix on ice during use. Aliquot and store at -20°C if they will not all be used at once. Avoid repeated freeze/thaw cycles. Use within two months. PC STANDARD: Dissolve in 200 µl dH2O to generate 50 mM (50 nmol/µl) PC Standard solution. Keep on ice while in use. Store at -20°C. Additional Materials Required •
Microcentrifuge
•
Pipettes and pipette tips
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Fluorescent or colorimetric microplate reader
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96 well plate
•
Orbital shaker
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4. Assay Protocol 1. Sample Preparation: Add samples to sample wells in a 96-well plate and bring the volume to 50 µl/well with Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute 10 µl of the 50 mM PC Standard with 990 µl dH2O to generate 0.5 mM standard Phosphatidylcholine. Add 0, 2, 4, 6, 8, 10 µl of the diluted PC Standard into a 96-well plate to generate 0, 1, 2, 3, 4, 5 nmol/well standard. Bring the volume to 50 µl with Assay Buffer. b. For the fluorometric assay: Dilute the standard to 0.05 mM (0.05 nmol/µl), then follow the same protocol as colorimetric assay. To generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol/well of the standard.
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3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare 50 µl Reaction Mix containing: Phosphatidylcholine
Background
Measurement
Control
Assay Buffer
44 µl
48 µl
PC Hydrolysis Enzyme
2 µl
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Development Mix
2 µl
2 µl
PC Probe**
2 µl
2 µl
4. Add 50 µl of the Reaction Mix to each well containing the PC standard and test samples. Mix well. Incubate the reaction for 30 min at room temperature, protect from light. * Note: Choline can generate significant background. If choline is present in your samples, perform a background control without the PC Hydrolysis Enzyme and subtract this value from sample readings. ** Note: For the fluorescent assay, dilute the probe 10X to reduce background reading. 5.
Measure
OD
at
570
nm
for
colorimetric
assay
(or
Ex/Em = 535/587 nm for fluorometric assay) in a microplate reader
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5. Data Analysis Correct background by subtracting the value derived from the zero PC control from all sample and standard readings. The background reading can be significant and must be subtracted from sample readings. Plot PC standard curve. Apply sample readings to the standard curve. PC concentrations of the test samples can then be calculated: Concentration = Sa / Sv (nmol/µl, or mM) Where: Sa is the PC content of unknown samples (in nmol) from standard curve. Sv is sample volume (µl) added into the assay wells. Phosphatidylcholine average molecular weight is 768 g/mol.
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Standard curves generated according to assay procedure.
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6. Troubleshooting Problem
Reason
Solution
Assay not working
Assay buffer at wrong temperature
Assay buffer must not be chilled - needs to be at RT
Protocol step missed Plate read at incorrect wavelength
Unsuitable microtiter plate for assay
Unexpected results
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
Measured at wrong wavelength
Use appropriate reader and filter settings described in datasheet
Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range
Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range
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Samples with inconsistent readings
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
Lower/ Higher readings in samples and standards
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)
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Problem
Reason
Solution
Standard curve is not linear
Not fully thawed kit components
Wait for components to thaw completely and gently mix prior use
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
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 (
[email protected]) or phone (select “contact us” on www.abcam.com for the phone number for your region).
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