Biology Reference
In-Depth Information
Table 1
Preparation of MDA Standards
Concentration
Third dilution
dH
2
O
0.1 μ
M
1.23 μL
498.8 μL
1.0 μ
M
12.3 μL
481.7 μL
5.0 μ
M
61.7 μL
438.3 μL
10 μ
M
123 μL
377
μL
3. Prepare MDA standards: Dilute the stock MDA by performing three 100-fold
serial dilutions in methanol (
see
Table 1
).
4. Combine 50 μL of sample/standard, 150 μL of 0.1
N
PCA, and 150 μL of 40 m
M
TBA in a 1.5-mL tube.
5. Vortex and incubate at 97 °C for 60 min in a fume hood.
6. Incubate at -20 °C for 20 min to cool the reaction.
7. Add 300 μL of methanol and 100 μL of 20% TCA.
8. Vortex and centrifuge at 13,000 ×
g
for 6 min.
9. Inject 10 μL onto C18 4.6 × 150-mm column with mobile phase 72:17:11 and
elute isocratically at 1 mL/min over 10 min. Monitor absorbance at 532 nm.
Acknowledgments
This research was supported by the Intramural Research Program of the
National Institutes of Health, National Institute of Allergy and Infectious
Diseases.
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