Aug 14, 2019

Public workspaceTRAP Assay

DOI
dx.doi.org/10.17504/protocols.io.3sqgndw
  • 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.3sqgndw
External link: https://www.diacomp.org/shared/document.aspx?id=32&docType=Protocol
Protocol Citation: Eva Feldman 2019. TRAP Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3sqgndw
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 14, 2019
Protocol Integer ID: 24112
Keywords: TRAP, cardiovascular, retinopathy, neuropathy, nephropathy, pediatric endocrinology, uropathy, wound-healing
Abstract
Summary:

The use of total radical-trapping antioxidant parameter (TRAP) has recently been proposed to explore the antioxidant property of a plasma sample. This assay is a measure of oxidative stress in the animals. This protocol describes the procedure used by the DiaComp to measure TRAP.





Diabetic Complications:








Materials
MATERIALS
Reagent 100 mM ABAP Merck MilliporeSigma (Sigma-Aldrich)Catalog #44,091-4
Reagent30 mM PB pH 7.0
ReagentLuminol Amersham plcCatalog #RPN2106
Reagent Preparation:

100mM ABAP*: Add 54.24 mg to 2 mL 30 mM PB.

Luminol: Mix 2 reagent ½ & ½..


Before start
IMPORTANT: Be sure light shield is in place

Sample Preparation - DRG:
Sample Preparation - DRG:
1. Remove 2 DRG from vial, cut in half and weigh. Do in duplicate.

2. Add 30 µL 30mM PB and sonicate on 4 on ice.

3. Spin at maximum g’s for 10 min at 4ºC.

4. Remove sup and store on ice.

Performing the Assay:
Performing the Assay:
1. Using a White Solid Bottom plate, prepare plate by loading buffer for serial dilution. No PB in 1st well, 5 µL PB in following 4 wells and 5 µL in 3 control well. (Control is PB, Luminol and ABAP) (Dilutions, 1:1, 1:2, 1:4, 1:8, 1:16)

2. Prepare ABAP just prior to running assay by dissolving 54.2 mg ABAP in 2 mL PB. (Enough for 3 columns)

3. Load 5 µL sample in wells 1 & 2. Mix the sample & PB in well 2 and remove 5 µL and place in 3rd well with PB and so on. On last dilution discard 5 µL.

4. Prepare Luminol in a 50 mL conical tube and add 200 µL per well.

5. Place plate in Fluoroskan and add 60 µL ABAP per well and press START.

6. Read every 30 seconds for 20 minutes.

7. When reading is done, Select Process>Organize. Choose the appropriate data to organize (usually Measure1), then click OK. This rearranges the data into columns.

8. Save organized data as an Excel file into the TRAP Assay data folder. Use the naming convention trXXXXXX.xls, where XXXXXX is the date in yymmdd format.


*ABAP = 2,2’-azobis(amidinopropane) dihydrochloride
*TRAP = Total Radical-trapping Antioxidant Parameter