Table 1.
Protocol for the antioxidant assay.
| Solution | Blanka (μL) | Sample (μL) |
|---|---|---|
| PBS (5 mM, pH 7.4, 138 mM NaCl) | 1000 – (105 + x) | 1000 – (115 + x) |
| MetMb (as determined above) | x b | x |
| Sample (serum or antioxidant) | — | 10 |
| ABTS (5 mM) | 30 | 30 |
| Equilibrate for 3 min at 30°C | ||
| H2O2 (10 mM) | 75 | 75 |
| Incubate for 6 min at 30°C | ||
| Read absorbance at 734 nm after exactly 15 s of incubation period completionc |
a Read against PBS buffer.
b MeMb concentration is determined experimentally and the required volume (x) is added to obtain 2.5 μM final concentration in the assay. The volume of PBS buffer is adjusted accordingly.
c Only six samples can be run at a time to fit the timing protocol.