Aug 09, 2019

Public workspaceThiobarbituric acid reactive substances (TBARS) Assay

DOI
dx.doi.org/10.17504/protocols.io.3sngnde
  • Eva Feldman1
  • 1University of Michigan - Ann Arbor
  • Diabetic Complications Consortium
    Tech. support email: rmcindoe@augusta.edu
Lili Liang
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DOI: dx.doi.org/10.17504/protocols.io.3sngnde
External link: https://www.diacomp.org/shared/document.aspx?id=33&docType=Protocol
Protocol Citation: Eva Feldman 2019. Thiobarbituric acid reactive substances (TBARS) Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3sngnde
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2019
Last Modified: August 09, 2019
Protocol Integer ID: 24110
Keywords: Biochemical Measures of Neuropathy, diabetic neuropathy, TBARS
Abstract
Summary:

Plasma concentrations of thiobarbituric acid reactive substances (TBARS) are an index of lipid peroxidation and oxidative stress. The protocol describes how the DiaComp quantitates TBARS in the animal models.



Diabetic Complication:


Materials
MATERIALS
ReagentThiobarbituric Acid (TBA)Catalog #ICN 190284
ReagentTrichloroacetic Acid Merck MilliporeSigma (Sigma-Aldrich)Catalog #490-10
Reagent1133-tetramethoxypropaneAcros OrganicsCatalog #148611000
Reagent Preparation:

Thiobarbituric Acid (TBA): 67 mg in 1mL DMSO then add 9 mL H²O.

10% Trichloroacetic Acid (w/v): in H²O.

1,1,3,3-tetramethoxypropane: 4.167 µL in 1mL Ethanol then add 49 mL H²O. (500 µM)



Note:

Sigma-Aldrich RRID:SCR_008988

Sample Preparation:
Sample Preparation:
Plasma:

• Place 100µL plasma into a labeled 1.5mL micro-centrifuge tube.

Tissue:

• Label 1 sets of 1.5mL micro-centrifuge tubes, 1 set screw top tubes and 1 set of 0.5mL tubes.

• Weighed out ~20mg and sonicate in 200µL RIPA buffer + inhibitors.

• Sonicate.

• Centrifuged @ 3000 for 10 min @ 4º .

1. Remove 10 µL aliquot into the 0.5mL tubes for protein analysis.

2. Place 100 µL lysate into a labeled 1.5mL micro-centrifuge tube.

3. Add 200µL ice cold 10% Trichloroacetic acid to precipitate protein.

4. Incubate for 15 minutes on ice.

5. Prepare standards as follows:


6. Centrifuge samples @ 2200 x g for 15 min. at @ 4ºC.

7. Place 200µL supernatant and standards into new labeled screw top 1.5ml tube.

8. Add and equal volume of 0.67% (w/v) TBA.

9. Incubate in a boiling water bath for 10 min.

10. Cool. Sample is ready for assay.

Performing Assay:
Performing Assay:
1. While samples are cooling, layout on computer and save as TBxxxxxx.sed where xxxxxx is the date in yyddmm format.

2. Load 150 µL into each standard well in duplicate.

3. Load 150 µL into each samples well in duplicate.

4. Put in plate reader and press start.
Reading the Plate:
Reading the Plate:
• Record absorbance at 532 nm.

1. Turn on Multiskan and open your saved file TBxxxxxx.sed.

2. Place plate onto Multiskan holder and click START.

3. Select Process>Curve Fit. Choose the appropriate data (usually Measure1), then click OK.

4. Save Curve Fit data sheet as an Excel file into the Data folder/TBARS data folder. Use the naming convention TBxxxxxx.xls, where xxxxxx is the date in yymmdd format.