This document provides instructions for using a lipid peroxidation assay kit to quantify malondialdehyde (MDA) levels as a marker of oxidative stress. The kit uses a colorimetric/fluorometric reaction between MDA and thiobarbituric acid to generate a product proportional to MDA levels. The procedure involves generating MDA standards, preparing samples in lysis buffer, reacting samples with thiobarbituric acid at high temperature, and measuring the resulting product by fluorescence or absorbance to quantify MDA levels based on a standard curve. Precautions are provided to ensure accurate and reproducible results.
This document provides instructions for using a lipid peroxidation assay kit to quantify malondialdehyde (MDA) levels as a marker of oxidative stress. The kit uses a colorimetric/fluorometric reaction between MDA and thiobarbituric acid to generate a product proportional to MDA levels. The procedure involves generating MDA standards, preparing samples in lysis buffer, reacting samples with thiobarbituric acid at high temperature, and measuring the resulting product by fluorescence or absorbance to quantify MDA levels based on a standard curve. Precautions are provided to ensure accurate and reproducible results.
This document provides instructions for using a lipid peroxidation assay kit to quantify malondialdehyde (MDA) levels as a marker of oxidative stress. The kit uses a colorimetric/fluorometric reaction between MDA and thiobarbituric acid to generate a product proportional to MDA levels. The procedure involves generating MDA standards, preparing samples in lysis buffer, reacting samples with thiobarbituric acid at high temperature, and measuring the resulting product by fluorescence or absorbance to quantify MDA levels based on a standard curve. Precautions are provided to ensure accurate and reproducible results.
This document provides instructions for using a lipid peroxidation assay kit to quantify malondialdehyde (MDA) levels as a marker of oxidative stress. The kit uses a colorimetric/fluorometric reaction between MDA and thiobarbituric acid to generate a product proportional to MDA levels. The procedure involves generating MDA standards, preparing samples in lysis buffer, reacting samples with thiobarbituric acid at high temperature, and measuring the resulting product by fluorescence or absorbance to quantify MDA levels based on a standard curve. Precautions are provided to ensure accurate and reproducible results.
Product Description Reagents and Equipment Required but Not
Lipid peroxidation is the degradation of lipids that Provided. occurs as a result of oxidative damage and is a useful • 96 well flat-bottom plate – It is recommended to use marker for oxidative stress. Polyunsaturated lipids are black plates with clear bottoms for fluorescence susceptible to an oxidative attack, typically by reactive assays and clear plates for colorimetric assays. oxygen species, resulting in a well-defined chain • Fluorescence or spectrophotometric multiwell plate reaction with the production of end products such as reader. malondialdehyde (MDA). Lipid peroxidation may • Glacial acetic acid (Catalog Number A6283 or contribute to the pathology of many diseases including equivalent) atherosclerosis, diabetes, and Alzheimer’s. • Sulfuric acid (Catalog Number 258105 or equivalent) In this kit, lipid peroxidation is determined by the • n-Butanol (Catalog Number 360465 or equivalent) reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric (532 nm)/fluorometric (λex = 532/ Precautions and Disclaimer λem = 553 nm) product, proportional to the MDA This product is for R&D use only, not for drug, present. household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards Components and safe handling practices. The kit is sufficient for 100 assays in 96 well plates. Preparation Instructions MDA Lysis Buffer 25 mL Briefly centrifuge vials before opening. To maintain Catalog Number MAK085A reagent integrity, avoid repeated freeze/thaw cycles. Use ultrapure water for the preparation of all reagents. Phosphotungstic Acid Solution 12.5 mL Allow all components to come to room temperature Catalog Number MAK085B before starting.
BHT, 100× 1 mL TBA Solution – Reconstitute a bottle with 7.5 mL
Catalog Number MAK085C Glacial Acetic Acid, then adjust the final volume to 25 mL with water. Sonication can be used to assist TBA 4 bottles dissolution if necessary. Store at room temperature Catalog Number MAK085D and use within 1 week of preparation.
MDA Standard, 4.17 M 0.1 mL Storage/Stability
Catalog Number MAK085E The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended. 2
Procedure Assay Reaction
All samples and standards should be run in duplicate. 1. To form the MDA-TBA adduct, add 600 µL of the TBA solution into each vial containing standard and MDA Standards for Colorimetric Detection sample. Incubate at 95 °C for 60 minutes. Cool to Dilute 10 µL of the 4.17 M MDA Standard Solution with room temperature in an ice bath for 10 minutes. 407 µL of water to prepare a 0.1 M MDA Standard Pipette 200 µL from each reaction mixture into a Solution. Further dilute 20 µL of the 0.1 M MDA 96 well plate for analysis. lo que pongo en la microplaca
Standard Solution with 980 µL of water to prepare a Notes: Occasional samples will exhibit turbidity, 2 mM MDA Standard. Add 0, 2, 4, 6, 8, and 10 µL of which can be eliminated by filtering through a the 2 mM MDA Standard Solution into separate 0.2 µm filter. TBA can react with other compounds microcentrifuge tubes, generating 0 (blank), 4, 8, 12, in samples giving other colored products. These 16, and 20 nmole standards. Add water to each tube to should not generally interfere with quantitation of bring the volume to 200 µL. the TBA-MDA adduct.
MDA Standards Fluorometric Detection To enhance sensitivity, 300 µL of n-butanol can be
Prepare a 2 mM Standard Solution as for the added to the 800 µL reaction mixture. If separation colorimetric assay. Take 100 µL of the 2 mM MDA does not occur, add 100 µL of 5 M NaCl and vortex Standard Solution and add to 900 µL of water to make vigorously. Centrifuge at 16,000 × g for 3 minutes a 0.2 mM MDA standard solution. Add 0, 2, 4, 6, 8, and to separate the layers. Transfer the n-butanol 10 µL of the 0.2 mM MDA standard solution into (upper) layer to a new tube, evaporate the separate microcentrifuge tubes, generating 0 (blank), n-butanol, dissolve the MDA-TBA adduct in 200 µL 0.4, 0.8, 1.2, 1.6, and 2.0 nmole standards. Add water of water, and then transfer to a 96 well plate for to each tube to bring the volume to 200 µL. analysis.
Sample Preparation 2. For colorimetric assays, measure the absorbance
Serum or Plasma samples (10 µL) should be gently at 532 nm (A532). For fluorometric assays, measure mixed with 500 µL of 42 mM H2SO4 in a microcentrifuge fluorescence intensity (λex = 532/λem = 553 nm). tube. Add 125 µL of Phosphotungstic Acid Solution and mix by vortexing. Incubate at room temperature for 5 minutes and then centrifuge the samples at 13,000 × g for 3 minutes. In a separate tube, add 2 µL of BHT (100×) to 100 µL of water. Resuspend the pellet on ice with the water/BHT solution. Adjust the volume to 200 µL with water.
Tissue (10 mg) or cells (1 × 106) can be homogenized
on ice in 300 µL of the MDA Lysis Buffer containing 3 µL of BHT (100×). Centrifuge the samples at 13,000 × g for 10 minutes to remove insoluble material. Alternatively, protein can be precipitated by homogenizing 10 mg of sample in 150 µL of the MDA Lysis Buffer containing 3 µL of BHT (100×) and adding 1 volume of 2 N perchloric acid, vortexing, and centrifuging to remove precipitated protein. Place 200 µL of the supernatant from each homogenized sample into a microcentrifuge tube. 3
Results Calculations Concentration of MDA The background for either assay is the value obtained for the 0 (blank) MDA standard. Correct for the (Sa/Sv) × 4 × D = C background by subtracting the blank value from all readings. Background values can be significant and Sa = Amount of MDA in unknown sample (nmole) from must be subtracted from all readings. Use the values standard curve obtained from the appropriate MDA standards to plot a Sv = Sample volume (µL) or amount (µg) added into the standard curve. The amount of MDA present in the wells samples may be determined from the standard curve. C = Concentration of MDA in sample Note: A new standard curve must be set up each time D = Dilution factor the assay is run. 4 = Correction factor for using 200 µL of 800 µL reaction
Sample Calculation Amount of MDA (Sa) = 5.84 nmole Sample volume (Sv) = 50 µL Concentration of MDA in sample
(5.84 nmole/50 µL) × 4 × 1 = 0.4672 nmole/µL
4
Troubleshooting Guide
Problem Possible Cause Suggested Solution
Cold assay buffer Assay Buffer must be at room temperature Omission of step in procedure Refer and follow Technical Bulletin precisely Plate reader at incorrect wavelength Check filter settings of instrument Assay not working For fluorescence assays, use black plates Type of 96 well plate used with clear bottoms. For colorimetric assays, use clear plates Use the Assay Buffer provided or refer to Samples prepared in different buffer Technical Bulletin for instructions Repeat the sample homogenization, Cell/Tissue culture samples were increasing the length and extent of incompletely homogenized homogenization step. Samples with erratic Samples used after multiple freeze-thaw Aliquot and freeze samples if samples will be readings cycles used multiple times Presence of interfering substance in the If possible, dilute sample further sample Use of old or inappropriately stored Use fresh samples and store correctly until samples use Thaw all components completely and mix Improperly thawed components gently before use Use of expired kit or improperly stored Check the expiration date and store the Lower/higher reagents components appropriately readings in samples Allowing the reagents to sit for extended Prepare fresh reaction mix before use and standards times on ice Refer to Technical Bulletin and verify correct Incorrect incubation times or temperatures incubation times and temperatures Incorrect volumes used Use calibrated pipettes and aliquot correctly Thaw and resuspend all components before Use of partially thawed components preparing the reaction mix Pipetting errors in preparation of standards Avoid pipetting small volumes Prepare a master Reaction Mix whenever Pipetting errors in the Reaction Mix possible Non-linear standard Pipette gently against the wall of the plate Air bubbles formed in well curve well Standard stock is at incorrect Refer to the standard dilution instructions in concentration the Technical Bulletin Recheck calculations after referring to Calculation errors Technical Bulletin Substituting reagents from older kits/lots Use fresh components from the same kit Samples measured at incorrect Check the equipment and filter settings wavelength Unanticipated results Samples contain interfering substances If possible, dilute sample further Sample readings above/below the linear Concentrate or dilute samples so readings range are in the linear range
LS,MAM 02/14-1
2014 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered i the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.
