I'm planning to perform dpph antioxidant assay on my active compounds and I already have the protocol for that. I'm aware that i should include a blank and a positive control but I have zero idea how to place them in the wells. like how many wells of blank and how many wells of controls should I fill? I have 2 samples each with 4 concentrations.
"Start by filling rows 1, 2, 3, and 4 with four different concentrations of your sample. Leave wells 5 empty and fill wells 6 and 7 with your positive and negative controls, respectively. Repeat this sequence in triplicate in columns A, B, and C. Then, leave the next 2column blank and repeat the same procedure with the next sample."
Hello,
I will mainly talk to you about the controls.
For the positive control, you can place it in the first column, it contains DPPH + ascorbic acid (reference antioxidant).
For the negative controls, there are 2. The first negative control to be placed in the second column, it only contains the DPPH and not your sample, and the second negative control to be placed in the third column, it only contains your sample and not the DPPH.
From column 4, deposit your samples with the different concentrations.
For free radical scavenging activity assay for 2, 2-diphenyl-1-picrylhydrazyl (DPPH), For the positive control, you can put it in the first well (DPPH+ascorbic acid). You can set the negative controls in two different wells or two negative controls you can use (As Sofiane Benyamina said earlier). The first negative control is to be placed in the second well (only DPPH), and the second negative control is to be placed in the third well (only sample). From the fourth well onwards you can put your samples in a triplicate manner.
Finally, the scavenging potential of DPPH radical was calculated as a % inhibition formula using the following equation.
% inhibition= [1- (As – Ab)/Ac] × 100
Mohd Akram Ansari Thank you so much for your answer. For the second negative control, can I choose any concentration from my samples? Also, I read that I have to include a blank which is only methanol or ethanol. Is it necessary?
Rouaa Shaheen, for your new question about choose any concentration from your samples to have second negative control. no, if you are going to measure the DO, each concentration of your sample must have its own blank.
Rouaa Shaheen, whatever the concentrations you are using for your assessment of antioxidant potential same concentrations you can used for the second negative control as well. Additionally it is very necessary to have blank (DPPH+Methanol) or (DPPH+Ethanol).
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