Sample Preparation and Protein Carbonylation Measurement in Larvae

Larvae were weighed and homogenized in phosphate buffered saline (PBS) buffer to prepare samples for the analysis of protein carbonylation (PCO), acetylcholinesterase (AchE), catalase (CAT), malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione (GSH). The homogenate was centrifuged to collect the supernatant. PCO content was measured using the 2,4-dinitrophenylhydrazine (DNPH) method. Streptomycin sulfate was added to the supernatant prior to reacting with DNPH. The resulting precipitate was washed with an ethanol:ethyl acetate mixture followed by guanidine hydrochloride to remove unbound DNPH. The carbonyl and protein contents were then determined spectrophotometrically. Finally, the carbonyl content per unit of protein was calculated based on the protein content.