Frozen samples were thawed on ice and centrifuged at 20,000 g for 15 min at 4°C to pellet organs and any suspended bacteria. The supernatant was decanted and 50–100 μl of 0.25% Rapigest surfactant (Waters, Milford, MA, USA) in 50 mM ammonium bicarbonate buffer was added. Organs were subsequently disrupted by sonication on a Vibra-Cell VCX 750 machine (Sonics & Materials Inc., CT, USA) equipped with a cup-horn probe: 3 bursts at full power for 30 s each and incubated at 60°C for 30 min to complete tissue lysis.
Total protein content of each sample was determined by the Pierce Bicinchoninic Acid protein assay kit (Product #23227, Thermo Scientific, IL, USA) on a NanoDrop 2000 spectrophotometer (Thermo, MA, USA) according to the manufacturer’s instructions. All samples were measured in triplicate. Calibration curves were generated with Bovine Serum Albumin (BSA, Sigma, MO, USA).
Total protein content of each sample was determined by the Pierce Bicinchoninic Acid protein assay kit (Product #23227, Thermo Scientific, IL, USA) on a NanoDrop 2000 spectrophotometer (Thermo, MA, USA) according to the manufacturer’s instructions. All samples were measured in triplicate. Calibration curves were generated with Bovine Serum Albumin (BSA, Sigma, MO, USA).