Pierce bca protein assay reagents a and b

Whole cell extracts were prepared by harvesting cells by using 1×trypin-EDTA. Once detached, medium was added to inactivate the trypsin and samples were spun in 2000× rpm for 5 min. The supernatant was discarded and cell pellets were washed in PBS and spun for an additional 5 min (1200× g). The supernatant was discarded and the cell pellet was frozen at −80 °C until lysis was performed. Frozen pellets were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 5 mM Dithiothreitol (DTT), 1% NP-40 (or IGEPAL) and 1 tablet of protease inhibitors (“Complete” protease inhibitor mixture, as instructed by the manufacturer, Roche Applied Science; Burgess Hill, UK) supplemented with phosphatase inhibitors (1 mM sodium fluoride (NaF), 1 mM sodium orthovanadate (Na₃VO₄) and 1 mM PMSF as protease inhibitor. The samples were kept on ice for 10 min, vortexing every 3 min to disrupt the cell membranes. The supernatant (protein extract) was finally collected by centrifugation at 800× g for 10 min at 4 °C. Protein concentration was determined using the Pierce BCA Protein Assay Reagents A and B (Thermo Scientific) according to manufacturer’s instruction. Absorbance was read at 562 nm using the Sunrise spectrophotometer (Tecan; Reading, UK). Protein concentrations were determined by the equation of absorbance × 25 = µg/µL.