TBARS Assay Kit

Item No. 10009055

www.caymanchem.com
Customer Service 800.364.9897
Technical Support 888.526.5351
1180 E. Ellsworth Rd · Ann Arbor, MI · USA
 TABLE OF CONTENTS GENERAL INFORMATION
GENERAL INFORMATION 3 Materials Supplied
Materials Supplied
4 Safety Data
4 Precautions
4 If You Have Problems Item Number Item Quantity
5 Storage and Stability
5 Materials Needed but Not Supplied 10009199 Thiobarbituric Acid 1 vial
5 Alternate Assay
10009200 TBA Acetic Acid 2 vials
INTRODUCTION 6 Background
7 About This Assay 10009201 TBA Sodium Hydroxide (10X) 1 vial

PRE-ASSAY PREPARATION 8 Reagent Preparation 10009202 TBA Malondialdehyde Standard 1 vial

9 Sample Preparation
10009203 TBA SDS Solution 1 vial
ASSAY PROTOCOL 11 Plate Set Up
13 Colorimetric Standard Preparation 400014 96-Well Solid Plate (Colorimetric Assay) 1 plate
14 Fluorometric Standard Preparation
400017 96-Well Solid Plate (black) 1 plate
15 Performing the Assay
400012 96-Well Cover Sheet 2 covers
ANALYSIS
16 Colorimetric Calculations
18 Fluorometric Calculations
19 Performance Characteristics If any of the items listed above are damaged or missing, please contact our
Customer Service department at (800) 364-9897 or (734) 971-3335. We cannot
RESOURCES 20 Interferences accept any returns without prior authorization.

!
21 Troubleshooting
21 References WARNING: THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR
22 Plate Template HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE.
23 Notes
23 Warranty and Limitation of Remedy

GENERAL INFORMATION 3
Safety Data Storage and Stability
This material should be considered hazardous until further information becomes This kit will perform as specified if stored at 4°C and used before the expiration
available. Do not ingest, inhale, get in eyes, on skin, or on clothing. Wash date indicated on the outside of the box.
thoroughly after handling. Before use, the user must review the complete Safety
Data Sheet, which has been sent via email to your institution. Materials Needed But Not Supplied
Precautions 1. A plate reader capable of measuring absorbance between 530-540 nm or a
fluorometer with the capacity to measure fluorescence using an excitation
Please read these instructions carefully before beginning this assay. wavelength of 530 nm and an emission wavelength of 550 nm
It is recommended to take appropriate precautions when using the kit reagents 2. Adjustable pipettes and a repeating pipettor
(i.e., lab coat, gloves, eye goggles, etc.), as some of them may be harmful. 3. A source of pure water; glass distilled water or HPLC-grade water is
The sodium hydroxide and acid solutions are corrosive and harmful if swallowed. acceptable
Contact with skin may cause burns. In case of contact with skin or eyes, rinse 4. Container sufficient to boil samples and standards
immediately with plenty of water for 15 minutes.
5. 5 ml polypropylene screw-cap centrifuge tubes (i.e., VWR Item No.
Care should be exercised when removing samples from boiling water. 16465-262)
6. Centrifuge capable of spinning 5 ml centrifuge tubes at 1,600 x g at 4°C
If You Have Problems
Technical Service Contact Information Alternate Assay
Phone: 888-526-5351 (USA and Canada only) or 734-975-3888 Cayman Chemical also offers an alternative assay kit for TBARS assessment (TBARS
Fax: 734-971-3640 (TCA Method) Assay Kit; Item No. 700870). While maintaining the reliability and
Email: techserv@caymanchem.com accuracy of the of the original assay, this assay offers the advantages of smaller
working volumes (1 ml vs. 4 ml) and improved sample processing resulting from
In order for our staff to assist you quickly and efficiently, please be ready to supply the formation of harder protein pellets during TCA precipitation.
the lot number of the kit (found on the outside of the box).

4 GENERAL INFORMATION GENERAL INFORMATION 5

 INTRODUCTION About This Assay
Cayman’s TBARS Assay Kit provides a simple, reproducible, and standardized tool
Background for assaying lipid peroxidation in plasma, serum, urine, tissue homogenates, and
Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation. cell lysates. The MDA-TBA adduct formed by the reaction of MDA and TBA under
Lipid peroxidation is a well-established mechanism of cellular injury in both plants high temperature (90-100°C) and acidic conditions is measured colorimetrically
and animals and is used as an indicator of oxidative stress in cells and tissues.1,2 at 530-540 nm or fluorometrically at an excitation wavelength of 530 nm and
Lipid peroxides, derived from PUFAs, are unstable and decompose to form a an emission wavelength of 550 nm. Although this reaction has a much higher
complex series of compounds, which include reactive carbonyl compounds, such sensitivity when measured fluorometrically, protocols for both methods are
as MDA. In human platelets, thromboxane synthase also catalyzes the conversion provided (see Figure 1 below).
of PGH2 to thromboxane A2, 12(S)-HHTrE, and MDA in a ratio of 1:1:1.3 O

The measurement of Thiobarbituric Acid Reactive Substances (TBARS) is a O O

N
S N OH HO N SH

well-established method for screening and monitoring lipid peroxidation.1,2

+ 2
+ 2 H 2O
H H N N
Modifications of the TBARS assay by many researchers have been used to O N S CH CH CH

evaluate several types of samples including human and animal tissues and fluids, OH OH

drugs, and foods.4-8 Even though there remains a controversy cited in literature MDA TBA MDA-TBA Adduct
regarding the specificity of TBARS toward compounds other than MDA, it still
remains the most widely employed assay used to determine lipid peroxidation.2 Figure 1.
If lipoprotein fractions are first acid precipitated from the sample, interfering
soluble TBARS are minimized, and the test becomes quite specific for lipid
peroxidation.2 Lipids with greater unsaturation will yield higher TBARS values.
It is recommended that if high TBARS values are obtained, a more specific assay
such as HPLC should be performed.

6 INTRODUCTION INTRODUCTION 7
 PRE-ASSAY PREPARATION Sample Preparation
Reagent Preparation Plasma
Typically, normal human plasma has a lipid peroxide level (expressed in terms of
1. Thiobarbituric Acid - (Item No. 10009199)
MDA) of 1.86-3.94 µM.1,8
The vial contains 2 g of thiobarbituric acid (TBA). It is ready to use to prepare
1. Collect blood using an anticoagulant such as heparin, EDTA, or citrate.
the Color Reagent.
2. Centrifuge the blood at 700-1,000 x g for 10 minutes at 4°C. Pipette off
2. TBA Acetic Acid - (Item No. 10009200) the top yellow plasma layer without disturbing the white buffy layer. Store
Each vial contains 20 ml of concentrated acetic acid. Slowly add both vials plasma on ice. If not assaying the same day, freeze at -80°C. The plasma
(40 ml) of TBA Acetic Acid to 160 ml of HPLC-grade water. This diluted sample will be stable for one month while stored at -80°C.
Acetic Acid Solution is used in preparing the Color Reagent. The diluted 3. Plasma does not need to be diluted before assaying.
Acetic Acid Solution is stable for at least three months at room temperature.
Serum
3. TBA Sodium Hydroxide (10X) - (Item No. 10009201)
Typically, normal human serum has a lipid peroxide level (expressed in terms of
The vial contains a solution of sodium hydroxide (NaOH). Dilute 20 ml of
MDA) of 1.86-3.94 µM.1
TBA NaOH with 180 ml of HPLC-grade water. This diluted NaOH Solution is
used in preparing the Color Reagent. The diluted NaOH Solution is stable for 1. Collect blood without using an anticoagulant.
at least three months at room temperature. Store the diluted NaOH Solution 2. Allow blood to clot for 30 minutes at 25°C.
in a plastic container suitable for corrosive materials.
3. Centrifuge the blood at 2,000 x g for 15 minutes at 4°C. Pipette off the top
4. TBA Malondialdehyde Standard - (Item No. 10009202) yellow serum layer without disturbing the white buffy layer. Store serum on
The vial contains 500 µM malondialdehyde (MDA) in water. It is ready to use ice. If not assaying the same day, freeze at -80°C. The serum sample will be
to prepare the standard curve. stable for one month while stored at -80°C.
5. TBA SDS Solution - (Item No. 10009203) 4. Serum does not need to be diluted before assaying.
The vial contains a solution of sodium dodecyl sulfate (SDS). The solution is Urine
ready to use as supplied. Typically, normal human urine has a lipid peroxide level (expressed in terms of
6. To prepare the Color Reagent: MDA) of 0.8-2 µmol/g creatinine.9,10
The following amount of Color Reagent is sufficient to evaluate 1. Urine does not require any special treatments. If not assaying the same day,
24 samples. Adjust the volumes accordingly if more or less samples are freeze at -80°C.
going to be assayed. Weigh 530 mg of TBA (Item No. 10009199) and add to
≥150 ml beaker containing 50 ml of diluted TBA Acetic Acid Solution. Add
50 ml of diluted TBA Sodium Hydroxide and mix until the TBA is completely
dissolved. The solution is stable for 24 hours.
8 PRE-ASSAY PREPARATION PRE-ASSAY PREPARATION 9
Tissue Homogenates
ASSAY PROTOCOL
1. Weigh out approximately 25 mg of tissue into a 1.5 ml centrifuge tube.
2. Add 250 μl of RIPA Buffer (prepared from Item No. 10010263) containing Plate Set Up
protease inhibitors of choice (see Interferences section on page 20).
There is no specific pattern for using the wells on the plate. A typical layout of
3. Homogenize or sonicate the tissue on ice. standards and samples to be measured in duplicate is shown below in Figure 2.
4. Centrifuge the tube at 1,600 x g for 10 minutes at 4°C. Use the supernatant We suggest you record the contents of each well on the template sheet provided
for analysis. Store supernatant on ice. If not assaying the same day, freeze (see page 22).
at -80°C. The sample will be stable for one month.
5. Tissue homogenates do not need to be diluted before assaying. 1 2 3 4 5 6 7 8 9 10 11 12
Cell Lysates A A A S1 S1 S9 S9 S17 S17 S25 S25 S33 S33

1. Collect 2 x 107 cells in 1 ml of cell culture medium or buffer of choice, such B B B S2 S2 S10 S10 S18 S18 S26 S26 S34 S34
as PBS.
2. Homogenize or sonicate the cells on ice. C C C S3 S3 S11 S11 S19 S19 S27 S27 S35 S35

3. Use the whole homogenate in the assay, being sure to use the culture D D D S4 S4 S12 S12 S20 S20 S28 S28 S36 S36
medium as a sample blank.
E E E S5 S5 S13 S13 S21 S21 S29 S29 S37 S37
4. Cell lysates do not need to be diluted before assaying.
F F F S6 S6 S14 S14 S22 S22 S30 S30 S38 S38

G G G S7 S7 S15 S15 S23 S23 S31 S31 S39 S39

H H H S8 S8 S16 S16 S24 S24 S32 S32 S40 S40

A-H = Standards
S1-S40 = Sample Wells

Figure 2. Sample plate format

10 PRE-ASSAY PREPARATION ASSAY PROTOCOL 11

 Pipetting Hints Colorimetric Standard Preparation
• It is recommended that an adjustable pipette be used to deliver Dilute 250 µl of the MDA Standard (Item No. 10009202) with 750 µl of water
reagents to the wells. to obtain a stock solution of 125 µM. Take eight clean glass test tubes and label
• Before pipetting each reagent, equilibrate the pipette tip in that them A-H. Add the amount of 125 µM MDA stock solution and water to each
reagent (i.e., slowly fill the tip and gently expel the contents, repeat tube as described in Table 1.
several times).
• Do not expose the pipette tip to the reagent(s) already in the well. Tube MDA (μl) Water (μl) MDA Concentration (μM)

General Information A 0 1,000 0

• All reagents except samples must be equilibrated to room temperature B 5 995 0.625
before beginning the assay. The SDS Solution will take at least one hour to
equilibrate to room temperature if stored at 2-8°C. Briefly heating the SDS C 10 990 1.25
Solution at 37°C will re-dissolve the precipitated SDS. The SDS Solution can
then be stored at room temperature. D 20 980 2.5
• The final volume of the assay is 150 µl in all wells.
E 40 960 5
• The assay is performed at room temperature.
F 80 920 10
• It is not necessary to use all the wells on the plate at one time.
• It is recommended that the samples and standards be assayed at least in G 200 800 25
duplicate.
• It is recommended that the samples and standards be kept at 4°C after H 400 600 50
preparation to increase sensitivity and reproducibility.
• Monitor the absorbance at 530-540 nm or read fluorescence at an excitation Table 1. MDA colorimetric standards
wavelength of 530 nm and an emission wavelength of 550 nm.

12 ASSAY PROTOCOL ASSAY PROTOCOL 13

Fluorometric Standard Preparation Performing the Assay
Dilute 25 µl of the MDA Standard (Item No. 10009202) with 975 µl of water to 1. Label vial caps with standard number or sample identification number.
obtain a stock solution of 12.5 µM. Take eight clean glass test tubes and label 2. Add 100 µl of sample or standard to appropriately labeled 5 ml vial.
them A-H. Add the amount of 12.5 µM MDA stock solution and water to each
tube as described in Table 2. 3. Add 100 µl of SDS Solution to vial and swirl to mix.
4. Add 4 ml of the Color Reagent forcefully down side of each vial.
Tube MDA (μl) Water (μl) MDA Concentration (μM) 5. Cap vials and place vials in foam or some other holder to keep the tubes
upright during boiling.
A 0 1,000 0 6. Add vials to vigorously boiling water. Boil vials for one hour.
B 5 995 0.0625 7. After one hour, immediately remove the vials and place in ice bath to stop
reaction. Incubate on ice for 10 minutes.
C 10 990 0.125 8. After 10 minutes, centrifuge the vials for 10 minutes at 1,600 x g at 4°C.
Vials may appear clear or cloudy. Cloudiness will clear upon warming to
D 20 980 0.25
room temperature.
E 40 960 0.5 9. Vials are stable at room temperature for 30 minutes.
10. Load 150 µl (in duplicate) from each vial to either the clear plate (colorimetric
F 80 920 1
version) or to the black plate (fluorometric version).
G 200 800 2.5 11. Read the absorbance at 530-540 nm or read fluorescence at an excitation
wavelength of 530 nm and an emission wavelength of 550 nm.
H 400 600 5

Table 2. MDA fluorometric standards

14 ASSAY PROTOCOL ASSAY PROTOCOL 15

 ANALYSIS 0.120

Colorimetric Calculations 0.100 y = 0.0022x + 0.0005

r2 = 0.9999
1. Calculate the average absorbance of each standard and sample.

Absorbance (532 nm)

2. Subtract the absorbance value of the standard A (0 µM) from itself and all 0.080

other values (both standards and samples). This is the corrected absorbance.
3. Plot the corrected absorbance values (from step 2 above) of each standard 0.060

as a function of MDA concentration (see Table 1, on page 13).

4. Calculate the values of MDA for each sample from the standard curve. An 0.040

example of the MDA standard curve is shown on page 17 in Figure 3.

0.020

MDA (µM) =
[ (Corrected absorbance) - (y-intercept)
Slope ] 0.000
0 10 20 30 40 50

MDA (µM)

Figure 3. MDA colorimetric standard curve

16 ANALYSIS ANALYSIS 17
Fluorometric Calculations 18,000

1. Calculate the average fluorescence of each standard and sample. 16,000 y = 3,238x + 210
r2 = 0.999
2. Subtract the fluorescence value of the standard A (0 µM) from itself

Fluorescence (Relative Units)

14,000
and all other values (both standards and samples). This is the corrected
fluorescence. 12,000

3. Plot the corrected fluorescence values (from step 2 above) of each standard 10,000
as a function of MDA concentration (see Table 2, on page 14).
8,000
4. Calculate the values of MDA for each sample from the standard curve. An
example of the MDA standard curve is shown on page 19 in Figure 4. 6,000

[ ]
4,000
(Corrected fluorescence) - (y-intercept)
MDA (µM) = 2,000
Slope
0
0 1 2 3 4 5 6

MDA (µM)

Figure 4. MDA fluorometric standard curve

Performance Characteristics
Precision:
When a series of ten human plasma and sixteen human urine samples were
assayed on the same day, the intra-assay coefficient of variation was 5.5%
and 7.6%, respectively. When a series of eight human plasma and sixteen
human urine samples were assayed on seven different days under the same
experimental conditions, the inter-assay coefficient of variation was 5.9% and
5.1%, respectively.
Assay Range:
Under the standardized conditions of the assay described in this booklet, the
dynamic range of the kit is 0-50 µM (Colorimetric) or 0-5 µM (Fluorometric)
(µM = µmole/liter = nmol/ml) MDA equivalents.

18 ANALYSIS ANALYSIS 19
 RESOURCES Troubleshooting

Interferences Problem Possible Causes Recommended Solutions

The following reagents were tested for interference in the assay. Erratic values; dispersion A. Poor pipetting/ A. Be careful not to splash
of duplicates/triplicates technique the contents of the wells
Reagent Will Interfere B. Bubble in the well(s) B. Carefully tap the side of
the plate with your finger
(Yes or No)
to remove bubbles
Borate (50 mM) No
HEPES (100 mM) No No MDA was detected in A. MDA concentration A. Process more tissue
Buffers: the sample was too low (50-100 mg)
Phosphate (100 mM) No B. The sample was too B. Harvest more cells
Tris (25 mM) No dilute (2 x 108) and re-assay

CHAPS (≤1%) No
The fluorometer The GAIN setting is too Reduce the GAIN and re-read
Detergents: Triton X-100 (≤1%) No exhibited ‘MAX’ values high
for the wells
Polysorbate 20 (≤1%) No
Antipain (≤0.1 mg/ml) No
Chymostatin (≤10 µg/ml) No References
Leupeptin (≤10 µg/ml) No
Protease 1. Yagi, K. Methods in Molecular Biology 108, 101-106 (1998).
Inhibitors/ PMSF (≤200 µM) No
Chelators: 2. Armstrong, D. and Browne, R. Free Radicals in Diagnostic Medicine 366, 43-58 (1994).
Trypsin (≤10 µg/ml) No
3. Wang, L.-H., Tsai, A., and Hsu, P.-Y. J. Biol. Chem. 276(18), 14737-14743 (2001).
EDTA (≤1 mM) No
4. Ohkawa, H., Ohishi, N., and Yagi, K. Anal. Biochem. 95, 351-358 (1979).
EGTA (≤1 mM) No
5. Dawn-Linsley, M., Ekinci, F.J., Ortiz, D., et al. J. Neurosci. Meth. 141, 219-222 (2005).
Sucrose (250 mM) Yes
Others: 6. Draper, H.H., Squires, E.J., Mahmoodi, H., et al. Free Radic. Biol. Med. 15, 353-363
Glycerol (≤10%) No (1993).
7. Scoccia, A.E., Molinuevo, M.S., McCarthy, A.D., et al. BMC Clinical Pathology 1, (2001).
8. Richard, M.-J., Portal, B., Meo, J., et al. Clin. Chem. 38(5), 704-709 (1992).
9. Jacob, R.A., Aiello, G.M., Stephensen, C.B., et al. J. Nutr. 133, 740-743 (2003).
10. Goulart, M., Batoréu, M.C., Rodrigues, A.S., et al. Mutagenesis 20(5), 311-315 (2005).

20 RESOURCES RESOURCES 21
 NOTES

1 2 3 4 5 6 7 8 9 10 11 12

Warranty and Limitation of Remedy

Buyer agrees to purchase the material subject to Cayman’s Terms and Conditions.
Complete Terms and Conditions including Warranty and Limitation of Liability
information can be found on our website.
This document is copyrighted. All rights are reserved. This document may not, in
whole or part, be copied, photocopied, reproduced, translated, or reduced to any
electronic medium or machine-readable form without prior consent, in writing,
from Cayman Chemical Company.
©01/12/2017, Cayman Chemical Company, Ann Arbor, MI, All rights reserved.
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Printed in U.S.A.

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