Lipid Peroxidation (MDA) Assay Kit

Transcription

1 ab Lipid Peroxidation (MDA) Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Lipid Peroxidation in various samples. This product is for research use only and is not intended for diagnostic use.

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3 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

4 1. Overview Quantification of lipid peroxidation is essential to assess oxidative stress in pathophysiological processes. Lipid peroxidation forms Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as natural bi-products. Measuring the end products of lipid peroxidation is one of the most widely accepted assays for oxidative damage. Abcam s Lipid Peroxidation Assay Kit provides a convenient tool for sensitive detection of the MDA in a variety of samples. The MDA in the sample is reacted with Thiobarbituric Acid (TBA) to generate the MDA-TBA adduct. The MDA-TBA adduct can be easily quantified colorimetrically (λ = 532 nm) or fluorometrically (Ex/Em = 532/553 nm). This assay detects MDA levels as low as 1 nmol/well colorimetrically and 0.1 nmol/well fluorometrically. 3

5 2. Protocol Summary Sample Preparation Standard Curve Preparation Add TBA Solution Measure Optical Density or Fluorescence 4

6 3. Components and Storage A. Kit Components Item Quantity MDA Lysis Buffer 25 ml Phosphotungstic Acid Solution 12.5 ml BHT (100X) 1 ml TBA 4 x bottles MDA Standard (4.17 M) 100 µl Store the kit at -20 C, protect from light. Allow all components to warm to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay. Store all reagents at -20 C between use. TBA SOLUTION: Reconstitute one vial of TBA with 7.5 ml Glacial Acetic Acid (not provided), then adjust the final volume to 25 ml with ddh 2 O. Store at room temperature. Must be used within 6-8 hr. 5

7 B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Colorimetric or fluorescent microplate reader 96-well plate Orbital shaker Glacial Acetic Acid 42 mm H 2 SO 4 n-butanol 6

8 4. Assay Protocol 1. Sample Preparation: a. For tissue and cell samples: 10 mg tissue or cells ( ) can be homogenized on ice in 300 µl of the MDA Lysis Buffer (with 3 µl BHT (100X), then centrifuged (13,000 x g, 10 min) to remove insoluble material. Place 200 µl of the supernatant from each homogenized sample into a microcentrifuge tube. b. For plasma samples: Gently mix 10 µl plasma with 500 µl of 42 mm H 2 SO 4 (not provided) in a microcentrifuge tube. Add 125 µl of Phosphotungstic Acid Solution and mix by vortexing. Incubate at room temperature for 5 min, then centrifuge for 3 min at 13,000 x g. Collect the pellet and resuspended on ice with 100 µl ddh 2 O (with 2 µl BHT (100X)). Adjust the final volume to 200 µl with ddh 2 O. 2. Standard Curve Preparation: Dilute 10 µl of the MDA standard with 407 µl of ddh 2 O to prepare a 0.1 M MDA solution, then dilute 20 µl of the 0.1 M MDA solution with 980 µl of ddh2o to prepare a 2 mm MDA Standard. 7

9 a. For the colorimetric assay: Add 0, 2, 4, 6, 8, 10 µl of the 2 mm MDA Standard into separate microcentrifuge tubes and adjust the final volume to 200 µl with ddh 2 O to generate 0, 4, 8, 12, 16 and 20 nmol Standard per well. b. For the fluorometric assay: Dilute the 2 mm MDA Standard 10-fold (10 µl + 90 µl ddh 2 O), then add 0, 2, 4, 6, 8, 10 µl of the 0.2 mm MDA Standard into separate microcentrifuge tubes and adjust the final volume to 200 µl with ddh 2 O to generate 0, 0.4, 0.8, 1.2, 1.6 and 2.0 nmol Standard per well. 3. Develop: Add 600 µl of TBA solution into each vial containing standard and sample. Incubate at 95 C for 60 min. Cool to room temperature in an ice bath for 10 min. Pipette 200 µl (from each 800 µl reaction mixture) into a 96-well microplate for analysis. Note: For more sensitivity, one can add 300 µl n-butanol (not provided in the kit) to extract the MDA-TBA adduct from the 800 µl reaction mixture. The layers can be separated by centrifugation (3 min at 16,000 x g). Evaporate the n-butanol and dissolve the MDA-TBA adduct in 200 µl ddh 2 O then place into the 96-well microplate for analysis. 8

10 4. Measure: a. For the colorimetric assay: Read the absorbance at 532 nm. b. For the fluorometric assay: Read supernatants (Ex/Em 532/553 nm). It is recommended to set the instrument sensitivity to high with a slit width of 5 nm. 5. Data Analysis Plot the MDA Standard Curve, then calculate the MDA amount in the test sample. Determine the sample amount of MDA equivalents in nmol by interpolation from the standard curve. Correct sample values for any other dilutions performed during specimen preparation. Concentration = [(A/(mg or ml) ] x 4 x D = nmol/ml or nmol/mg Where: A is the sample MDA amount from the standard curve (in nmol). mg is the original tissue amount used (e.g. 10 mg) ml is the original plasma volume used (e.g ml) 4 is the correction for using 200 µl of the 800 µl reaction mix D is the dilution factor (if any BEFORE original amount or volume) 9

11 MDA Standard Curve performed according to the Assay Protocol. 10

12 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

13 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

14 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 (technical@abcam.com) or phone (select contact us on for the phone number for your region). 13

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16 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 15