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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

Standard

Volume of

Volume

Final volume standard in well (µL)

End amount of MDA standard in well (nmol/well)

#

MDA

ddH 2 0 (µL)

Standard (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