Pierce bca protein assay kit

To investigate the protein expression in signaling pathways of A549 and H1299 cells with δT exposure, 1,000,000 cells were plated per 100-mm dish and incubated for 24 hours. The cells were then grown for 72 hours with treatment and control medium. Next, the cells were lysed in the cold 1× cell lysis buffer (Cell Signaling Technology) for 30 minutes on ice with 1× protease inhibitor (Cell Signaling Technology), and protein concentrations were calculated using a Pierce BCA protein assay kit. Subsequently, 50 mg of total cell lysates was mixed with same amounts of 4× Laemmli buffer (Bio-Rad Laboratories), and the samples were loaded onto a 10% SDS–polyacrylamide gel. The gel was electrophoretically transferred to a polyvinylidene difluoride (PVDF) transfer membrane (Whatman, Clifton, NJ, USA) using a transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol) after gel electrophoresis. The PVDF membranes were incubated for 2 hours at room temperature with 5% BSA in 1× Tris-buffered saline buffer containing 0.1% Tween (TBS-T) and then incubated overnight at 4°C with primary antibodies (1:1,000). The membranes were washed three times with TBS-T and incubated with the secondary antibodies (1:5,000) containing 2% BSA at room temperature for 2 hours. The signal strength was then measured in an image with Chemi-Doc™ XRS system (Bio-Rad Laboratories).