DPPH Anti Oxidant Assay / TEST & IC50 CALCULATION

DPPH Anti Oxidant Assay / TEST - IC50 Calculation

NOTE: FOR BLANK PLEASE CONSIDER METHANOL AS THE SOLVENT... IT'S BEEN A TYPO ERROR FROM US...

dpph assay,
dpph free radical scavenging assay,
dpph,
dpph antioxidant activity,
frap assay for antioxidant activity,
antioxidant activity by dpph method,
dpph spectrophotometric assay,
DPPH Radical Scavenging Method,
Antioxidant Activity Protocol by DPPH,

0:00 - Intro
0:26 - What is DPPH?
4:00 - Principle for DPPH Assay
12:03 -Observation for the Assay
14:15 - Notes to be observed
17:16 - Chemical reactions and Note
20:42 - sample extraction and solutions preparations
29:39 - Procedure
31:18 - Protocol and DPPH Scavenging Effect
37:50 - IC'50 explanation
41:25 - Linear Regression and IC'50 formula
49:00 - Graph and IC'50 calculation

Dilution of samples/standards- We have adopted the method of serial dilution. But, the concentration adopted as micrograms -
As you have observed in the slide of Preparation of solutions - the Preparation of standard ascorbic acid (for Reference) is taken and an 800μg/ml of stock solution is prepared.
Now, when you have to prepare 400μg, 200μg, 100μg, 50μg, 25μg and 12.5μg of dilutions for the 6 references.

Arrange 6 test tubes and keep the 1st test tube empty and followed by 5 Test-tubes; each with 0.5 ml of distilled water.

1. Now, pipette out 1ml of stock solution which is 800μg/ml of ascorbic acid into the first empty test tube. Avoid generating bubbles.
2. Pipette 0.5 mL of dilution 1 (from step 1) into 2nd test tube 0.5 mL of distilled water to create dilution 2. Mix well before continuing. Avoid generating bubbles.
3. Pipette 0.5 mL of dilution 2 (from step 2) into 3rd test tube 0.5 mL of distilled water to create dilution 3. Mix well before continuing. Avoid generating bubbles.
4. Pipette 0.5 mL of dilution 3 (from step 3) into 4th test tube 0.5 mL of distilled water to create dilution 4. Mix well before continuing. Avoid generating bubbles.
5. Pipette 0.5 mL of dilution 4 (from step 4) into 5th test tube 0.5 mL of distilled water to create dilution 5. Mix well before continuing. Avoid generating bubbles.
6. Pipette 0.5 mL of dilution 5 (from step 5) into 6th test tube 0.5 mL of distilled water to create dilution 6. Mix well before continuing.

Now, Each test tube will have 400μg, 200μg, 100μg, 50μg, 25μg and 12.5μg of ascorbic acid. Then make up the volumes with distilled water to 6,5,4,3,2 from the 1st to 5th test tube and the last test tube already be 1ml.

Note: In this same manner, you have to perform serial dilution for the dry samples. Hope I have cleared your doubts.

Important Note:
We have used two different control preparations to mark the difference that also comes under the control. Hence, we have noted the same values. And you don't try the same.
Note the control absorbance value for ASA acid and sample should be the same.