I want to measure MDA concentrations using the TBARS assay in in vitro cell lines. Currently, I am following the protocol as described by (Aguilar et al., 2022) in their paper "Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay".
They have used RIPA buffer for lysis, and then incubated the cell lysate supernatant for 24 hours. Also, they have used 8.1% SDS, 3.5M sodium acetate and 0.8% Thiobarbituric acid (TBA) for the actual TBARS assay.
However, other protocols for TBARS use Trichloroacetic acid (TCA) in various concentrations and TBA for the TBARS assay, and do not have 24 hour incubation.
My main confusion is regarding the 24 hour incubation in the protocol. Won't this change the MDA levels and give inaccurate results? What is the principle behind this 24 hour incubation? Another confusion is regarding the composition of the reagents of the TBARS assay. What is the difference between the two?
I would appreciate clarifications regarding these, as I need to decide to procced with the protocol I am following or should I change to the TCA + TBA protocol.
Thank you.
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