Micro bicinchoninic acid assay bca kit

Tissue samples (n = 3) were excised and transferred into new tubes containing tissue lysis buffer (1% SDS, 8 mol/L urea) and 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, MO, USA). Then, the lysates were homogenized for 4 min using a TissueLyser (CK1000, Thmorgan, Beijing, China) and incubated on ice for 30 min. The lysates were centrifuged at 12,000× g and 4 °C for 15 min, and the supernatants were collected. The concentration of proteins was quantified using a micro-bicinchoninic acid assay (BCA) kit (Thermo-Fisher, Waltham, MA, USA) and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels.

To confirm the size and quantify the amounts of purified proteins, a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The purified proteins were mixed with a reducing sample buffer containing SDS and β-mercaptoethanol (pH 6.8), prior to loading on a 10% SDS-PAGE gel. Through the electrophoresis, each sample was separated depending upon size. Then, the polyacrylamide gel was immersed in a Coomassie Blue staining solution for visualization. Micro bicinchoninic acid assay (BCA) kit (Thermo Fisher Scientific, United States) was used to quantify the purified proteins. To identify the purified proteins, a Western blot analysis was conducted. After the transfer of purified products on SDS-PAGE gel to a polyvinylidene difluoride (PVDF) membrane, proteins were labeled with an anti-T7-tag antibody (Abcam, United Kingdom) and an anti-rabbit HRP-conjugated antibody (Millipore, United States). Lumina Forte Western HRP substrate (Millipore) was used to visualize proteins on the G-Box Chemi XL system (Syngene, United Kingdom).