Dear Prashant Neupane , when doing an antioxidant assay with DPPH and ABTS you have first to perform your assays in triplicate in order to minimise the experimental variations, for the concentrations you have to be sure the concentration you are following is the final one (take in consideration the 50µl sample dilution on the DPPH or ABTS). to minimize the error that can be on the experiment it's better to make a Mother solution and prepare your dilutions in sequence (errors can occur when preparing each tube alone) if U do so you should have an approximatively linear curve (100% linear curve is impossible to obtain). contact me on private message if you need help with your protocol or interpretations.

best regards.

Dear Hicham Mechqoq, Thank you for the suggestions. The experiments were performed in triplicates and samples were serially diluted in a 96 well microplate.

I did obtain a standard curve for Ascorbic acid with r square 0.977 with DPPH reagent.

My real struggle is with ABTS assay because almost all literatures suggest dilution of ABTS reagent such that the absorbance of working solution is 0.7 at 735nm. This working solution produced an exponential curve for gallic acid at concentrations ranging from 50ug/ml-0.125ug/ml. The trend is linear at concentrations less than 6.25ug/ml. What should I do?

Thankyou in advance!

For ABTS we prefer to use trolox more than ascorbic acid, if you dont have then you ll have to work with ascorbic acid, if the curve is linear in less than 6.25ug/ml, then this have to be the higher value and dilutions have to be made before this one to draw your curve.