ab83377 Phosphatidylcholine Assay Kit

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Add samples to sample wells in a 96-well plate and bring the volume to 50 µl/well .... contact us by email ([email protected]) or phone (select. “contact us” ...
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



Fluorescent or colorimetric microplate reader



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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UK, EU and ROW Email: [email protected] Tel: +44 (0)1223 696000 www.abcam.com US, Canada and Latin America Email: [email protected] Tel: 888-77-ABCAM (22226) www.abcam.com China and Asia Pacific Email: [email protected] Tel: 108008523689 (中國聯通) www.abcam.cn Japan Email: [email protected] Tel: +81-(0)3-6231-0940 www.abcam.co.jp

Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. 15 All information / detail is correct at time of going to print.