DPPH Free Radical Scavenging Assay

DPPH Free Radical Scavenging Assay

biochemistry · beginner · ⏱ 1 hour

https://labgrimoire.com/protocols/dpph-free-radical-scavenging-assay/

Materials

• ☐ DPPH (2,2-diphenyl-1-picrylhydrazyl) (store protected from light)
• ☐ Methanol (solvent; ethanol may substitute)
• ☐ Vitamin C (ascorbic acid) (positive control)
• ☐ Sample extract: 0.5 mL per reaction (cuvette) or 100 µL (microplate)
• ☐ Volumetric flask: 50 mL
• ☐ Aluminum foil (for light protection)

Equipment

• ☐ UV-Vis spectrophotometer (517 nm wavelength)
• ☐ Microplate reader (517 nm; needed for microplate format only)
• ☐ 96-well microplate (flat-bottom; microplate format only)
• ☐ Cuvettes (cuvette format only)
• ☐ Micropipettes (adjustable, 10–1000 µL)

Procedure

1. ☐ Prepare DPPH working solution (5 min)

   Dissolve DPPH in methanol to make a stock solution. Transfer 3 mL stock into a 50 mL volumetric flask and bring to volume with methanol. Wrap in aluminum foil.

   Adjust absorbance of the working solution to 1.00 ± 0.200 at 517 nm.

2. ☐ Prepare Vitamin C standard curve (10 min)

   Prepare a two-fold serial dilution of Vitamin C: 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 µg/mL.

   These serve as the positive control to validate assay performance and calculate IC50.

3. ☐ Cuvette format: Mix DPPH with sample (2 min)

   Add 3 mL DPPH working solution to 0.5 mL sample extract in a cuvette. Mix well by gentle inversion.

   For blank: replace sample with 0.5 mL solvent (methanol).

4. ☐ Cuvette format: Incubate in the dark (30 min)

   Place the cuvette in the dark (drawer or foil-covered rack) for 30 minutes at room temperature.

5. ☐ Cuvette format: Read absorbance at 517 nm (2 min)

   Measure absorbance at 517 nm (OD517) using the UV-Vis spectrophotometer. Record values for sample and blank.

6. ☐ Microplate format: Add sample to wells (5 min)

   Pipette 100 µL of each sample (or standard/blank) into a flat-bottom 96-well plate. Use triplicates for each concentration.

7. ☐ Microplate format: Add DPPH and mix (5 min)

   Add 300 µL of 0.4 mM DPPH to each well using a multichannel pipette. Mix by gentle orbital shaking for 10 seconds.

8. ☐ Microplate format: Incubate and read (30 min)

   Incubate in the dark for 30 minutes. Read absorbance at 517 nm on the microplate reader.

9. ☐ Calculate scavenging activity

   Use the formula:

   Scavenging Activity (%) = (1 − ODsample / ODblank) × 100%

   Plot concentration vs. scavenging activity to determine IC50 (the concentration at which 50% of DPPH radicals are scavenged).