4.6. Liver tissue homogenization and total protein determination

Whole liver tissues were weighed to approximately 50–100 mg and were placed in ceramic bead tubes on ice. 1 ml of RIPA lysis buffer and 1% Halt protease and phosphatase inhibitors were added to each tube. Samples were homogenized using two 15 second pulses at 6,800 RPM using Precellys Evolution tissue homogenizer. On observing a homogenized lysate solution, bead tubes were centrifuged at 18000 x g for 15 minutes at 4°C using Eppendorf 5430R tabletop centrifuge. Supernatants were carefully transferred to new labelled tubes and stored on ice. Tissue lysates were diluted 25-fold and diluted lysate was used to determine total protein concentration in duplicate by BCA assay in a 96 well plate format. Target protein detection was performed by capillary electrophoresis with liver lysates normalized to 0.2 mg/ml protein concentration as described previously.