Author: Philip Bissiwu

Contact: pbissiwu17@students.desu.edu, 3029221589

August 8, 2018

MDA assay protocol

Reagent Preparation

1. TBA Solution:
• Reconstitute one vial of TBA in 7.5 mL Glacial Acetic Acid.
• Adjust the final volume to 25 mL with ddH2O
• Mix well to dissolve

Note: Store at 4°C. Reconstituted TBA solution is stable for 1 week.

2. Lysis solution
Mix MDA Lysis Buffer with BHT(100X) according to the table bellow
Reagent 1 sample (uL) 12 samples (uL)
MDA lysis buffer 300 3600
BHT 3 36
Total 302 3636

3. Standard preparation
• Prepare a 0.1 M MDA standard by diluting 10 μL 4.17 M MDA Standard in 407 μL of
ddH2O.
• Prepare a 2 mM MDA standard by diluting 10 μL 0.1 M MDA Standard in 490 μL of
ddH2O.
• Using 2 mM MDA standard, prepare standard curve dilution as described in the table in
a microplate or micro centrifuge tube
•

Volume of End amount of

Standard Final volume
MDA Volume MDA standard in
# standard in well
ddH20 (µL) well (nmol/well)
Standard (µL) (µL)

1 0 600 200 0
2 6 594 200 4 (20 µM)
3 12 588 200 8 (40 µM)
4 18 582 200 12 (60 µM)
5 24 576 200 16 (80 µM)
6 30 570 200 20 (100 µM)
Author: Philip Bissiwu
Contact: pbissiwu17@students.desu.edu, 3029221589
August 8, 2018

Assay procedure

1. Homogenize tissue in 303 μL Lysis Solution (Buffer + BHT)

2. Centrifuge at 13,000 x g for 10 minutes to remove insoluble material
3. Collect the supernatant.
4. Put 200 ul of the supernatant into centrifuge tubes
5. Put 200 ul of the standard into centrifuge tubes
6. Add 600 uL of TBA reagent to both the standards and the samples
7. Incubate at 95°C for 60 minutes
8. Cool to room temperature in an ice bath for 10 minutes
9. Take 200 ul of both the incubated standard and samples in 96 well plate for analysis
10. Measure absorbance immediately on a microplate reader at OD 532 nm

Data analysis
• Average the duplicate reading for each standard and sample.
• Subtract the mean value of the blank (Standard #1) from all standards and sample
readings. This is the corrected absorbance.
• Plot the corrected values for each standard as a function of the final concentration of
MDA.
• Draw the best smooth curve through these points to construct the standard curve
Calculate the trendline equation based on your standard curve data (use the equation
that provides the most accurate fit).
• Apply the corrected sample OD reading to the standard curve to get MDA amount (y =
mx + b) in the sample wells.
• Concentration of MDA in the test samples is calculated as:

MDA concentration = [ ( A/tissue weight (ml)] *4*D

Unit Expressed in nmol/mg

A = MDA calculated from the curve

D = Dilution factor
4 = correction factor for using 200ul of the 600 ul
Author: Philip Bissiwu
Contact: pbissiwu17@students.desu.edu, 3029221589
August 8, 2018

Notes