Bca protein assay kit

The protein levels were determined by the western blotting assay. Total protein lysis was prepared using the RIPA Lysis Buffer (P0013B, Beyotime) and quantified by the BCA Protein Assay Kit (T9300A, Takara). The protein samples for western blotting were prepared using SDS-PAGE Sample Loading Buffer (P0015L, Beyotime). Equal amounts of total proteins were loaded onto 10% SDS-PAGE (P0012AC, Beyotime) and separated by electrophoresis. The separated proteins were transferred onto a PVDF membrane (IPVH00010, Millipore, Shanghai, China) and blocked by 5% skim milk (232100, BD Bioscience, San Jose, CA, USA). The membranes were incubated with primary antibodies Bcl-2 Rabbit Polyclonal Antibody (1:1000, AF0060, Beyotime), Anti-Survivin Rabbit pAb (1:1000, GB11177, Servicebio, Wuhan, China) and GAPDH Mouse Monoclonal antibody (1:5000, 60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. Then HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:5000, SA00001-1, Proteintech) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:5000, SA00001-2, Proteintech) were used to probe the membrane at room temperature for 1 h. The protein bands were visualized using Amersham ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare, Princeton, NJ, USA) and imaged by Tanon-5200 chemiluminescence detection system (Tanon).

A-PRF/CGF/PPTF clots (1 mm thick) were compressed in the stainless-steel compressor [16 (link)] and were punched out (φ8 mm) using a biopsy punch (Kai Corp., Tokyo, Japan). After repeatedly rinsing the disks with PBS to eliminate as much serum as possible, the disks were immersed into 4 mL of 0.05% trypsin plus 0.53 mM EDTA (Invitrogen, Carlsbad, CA, USA) in a 35-mm dish inside a CO₂ incubator. Fibrin is well known to be specifically degraded by plasmin in vivo; however, because it takes a long time to determine degradation using plasmin in vitro [12 (link)] and because fibrin could be degraded also by other proteases in vivo, we used trypsin plus EDTA, which is usually used in cell culture, in this study.
After pipetting the digestion solution, 50 μL of the digestion solution was collected every 20 min and was stored at −20 °C until protein measurement. Protein levels, which can be considered primarily as levels of digested fibrin fiber, were then determined by a BCA protein assay kit (Takara Bio, Kusatsu, Japan). The protein levels at the time point when the initial fibrin disks were completely digested overnight were evaluated at 100%.

Total proteins were extracted from cells using ice‐cold Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitors. BCA Protein Assay Kit (Takara) was used to quantify protein concentration. Approximately 20 μg of total protein lysate was separated by 10% SDS‐polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were blocked with 5% skimmed milk for 2 hours and then incubated overnight with primary antibodies. After three rinses, membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies at room temperature for 1 hour. Signals were visualized with Enhanced Chemiluminescence Detection Kit (Pierce Biotechnology).

Cells were lysed in RIPA lysis buffer (Beyotime, China), and the total protein concentrations were detected using the BCA Protein Assay Kit (TaKaRa, Japan). The proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes. All membranes were blocked with 5% BSA. The membranes were incubated with a rabbit polyclonal anti-human collagen type I antibody (ab34710; Abcam, dilution 1 : 4000), a rabbit polyclonal anti-human collagen type III antibody (ab7778; Abcam, dilution 1 : 4000), an anti-alpha muscle actin antibody (ab5694; Abcam, dilution 1 : 1000), or β-actin (BS13278, Bioworld, dilution 1 : 1500) at 4°C overnight. Subsequently, each membrane was incubated with the corresponding secondary antibodies at room temperature for 1 h. Finally, we used the enhanced chemiluminescence kit to observe protein bands in a ChemiDoc XRS Plus c (Bio-Rad, USA).