Coomassie blue g 250 solution

Approximately 100 mg of epimastigote cell pellets were used for the extraction of proteins. The samples were homogenized in 500 μl rehydration solution (7 M urea, 2 M thiourea, 2% w/v CHAPS, 0.002% w/v bromophenol blue; all from Sigma-Aldrich, Missouri, USA) containing 1× Protease Inhibitor Cocktail (Sigma-Aldrich) at a final dilution of 1:25, for 10 min in an ice bath. The samples were then sonicated by means of a series of three 15 s pulses, each followed by 45 s resting on ice between cycles. Homogenates were centrifuged at 20,000× g for 1.5 h at 4 °C to remove cell debris. The 1D electrophoretic profiles were used to verify the integrity of the extracts and to normalize, through densitometric analysis (software Quantity one v.29.0), the total amount of protein present in each sample. Briefly, a 5 μl aliquot of each sample was separated under denaturing conditions using 12% SDS-PAGE. After 30 min in fixative solution (40% ethanol/7% acetic acid), the gel was stained in 0.02% Coomassie Blue G-250 solution (Sigma-Aldrich) for 2 h.

100 μL of hATT-CMs (n=7) and hATN-CMs (n=3) were loaded onto a 10% SDS-PAGE gel, and proteins were separated by electrophoresis, at a constant voltage of 100 V. After the run, the gel was fixed with a 50% v/v ethanol, 2% v/v phosphoric acid solution, overnight at 4°C. Then, gel was washed 3 times and proteins were dyed with Coomassie Blue G250 solution (Sigma Chemical Co, St. Louis, MO, USA). Control-CM was used as control and a lane with molecular weight markers was included.