Pierce bca protein assaykit assay

For protein precipitation
studies, a series of 10 mg/mL lysozyme samples and ATP or TPP concentrations
ranging from 0 to 100 mM in 10 mM Tris pH 7.0 were prepared. After
mixing, the samples were left to equilibrate at room temperature for
60 days. After 2 h of incubation, 50 μL of supernatant was collected,
centrifuged at 10,000 rpm for 10 min using a Heraeus Pico 17 Centrifuge
(Thermo Fisher Scientific Ltd., U.K.), and its protein concentration
measured. This step was repeated after 60 days of incubation at room
temperature. For samples containing TPP, the protein concentration
was determined by measuring the absorbance at 280 nm using a NanoDrop
2000 (Thermo Fisher Scientific Ltd., U.K). For samples containing
ATP, the concentration was measured using the Pierce BCA Protein Assay
Kit assay (Thermo Fisher Scientific Ltd., U.K.) per manufacturer’s
protocol. A stock solution of lysozyme with a known concentration
was used to prepare the dilution series of standard samples to obtain
the standard protein concentration curve.

Daoy and HD-MB03 cells were cultured to sub-confluence, then lysed on ice with 1X Laemmli buffer (6 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol). The lysates were sonicated for 30 s and protein amount was determined by the Pierce™ BCA Protein Assay Kit assay (Thermo Fisher). Sample proteins were reduced by heating at 95 °C for 5 min with 5% β-mercaptoethanol. 50 μg of proteins were loaded on 10% polyacrylamide gels and SDS-PAGE was performed using the Mini-Protean® Tetra Vertical Electrophoresis Cell (Biorad, Marnes-la-Coquette, FRANCE). The proteins were transferred onto PVDF membranes in Tris-Glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) + 20% ethanol (v:v) using the Hoefer wet blotting system (Thermo Fisher). Membranes were air-dried and blocked with 3% BSA, at room temperature for 1 h, then immunoblotted overnight with primary antibodies diluted in 3% BSA, at 4 °C. Membranes were washed with water, incubated with HRP-conjugated secondary antibodies, at room temperature, for 1 h. After final washing with water, the Advansta WesternBright Quantum HRP substrate (Diagomics, Blagnac, FRANCE) was used as detection reagent.