Bca protein concentration assay kit

Cells were harvested and lyzed in Radio-Immunoprecipitation Assay (RIPA) lysis buffer, and centrifuged at 4°C 12,000 g for 10 min. The concentration of each protein sample were measured using the BCA protein concentration assay kit (Wanleibio, Shenyang, China). Proteins (30 μg) extracted from cells were loaded and separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk and incubated with anti-SP1 (1:1000, 9389, Cell Signaling Technology, MA, USA) in 5% milk/PBS buffer at 4°C overnight, followed by incubation with goat anti-rabbit secondary antibody (A23720, Abbkine, Wuhan, China) conjugated with DyLight fluorescent 680. After extensive washing with PBST buffer, the protein band was visualized under an Odyssey CLx imaging system (LI-COR, Inc., NE, USA) [30 (link)].

Cells were lysed with Ripa buffer (Wanleibio, China) containing 10 mM protease inhibitor (PMSF; Wanleibio, China). The lysate was centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was separated, and the protein concentration was determined using a BCA protein concentration assay kit (Wanleibio, China). Equivalent amounts of protein (40 g) were added to the 8%-15% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, Billerica, United States). The membrane was sealed in 5% skimmed milk powder solution for 1 h, and then incubated with the following primary antibodies: runt-related transcription factor 2 (Runx2, wl03358; Wanleibio, China), Collagen I (wl0088; Wanleibio, China), β-actin (wl01845; Wanleibio, China), CD9 (ab92726, Abcam, United Kingdom), CD63 (ab216130, Abcam, United Kingdom), and TSG101 (ab125011, Abcam, United Kingdom) at 4°C overnight. After washing with TBST four times, the membrane was incubated with sheep anti-rabbit IgG HRP (wla023; Wanleibio, China) at room temperature for 45 min. An ECL detection kit (Wanleibio, China) was used to visualize the protein bands. Proteins on the same membrane were compared quantitatively by determining the optical density of the target strip using a gel image processing system (Gel-Pro-Analyzer software, Media Cybernetics, United States).