Bca protein kit

The total protein and membrane protein of cells were prepared with protein extraction reagent (Tiangen, Beijing, China) and a Membrane and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China), respectively. The protein levels were assayed by the BCA protein kit (Tiangen, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) filters membrane using a Mini-Protean 3 System (Bio-Rad, Hercules, CA, USA). The membrane was blocked in blocking buffer with 5% skim milk for 1 h and then incubated with specific primary polyclonal/monoclonal antibodies overnight at 4 °C. After incubation with horseradish peroxidase conjugated secondary antibody for 2 h, immunoreactive proteins were visualized with the ECL Plus Western Blotting Detection Reagents (Fude-bio, Hangzhou, China) and detected using a Mini-Protein System (Bio-Rad). Protein bands were quantitated with NIH ImageJ software and normalized by GAPDH bands for analysis.

Forty milliliters of overnight bacteria culture supernatants were obtained by centrifugation and filtration through a 0.22 μm filter. Proteins were precipitated through the addition of 3 volumes of ice-cold ethanol with the protein pellet resuspended in 500 μL of PBS. Equal loading was confirmed by measurement of total protein with the BCA protein Kit (Tiangen Biotech, Beijing, China). Proteins were separated by a 12% Tris·HCl SDS/PAGE gel (Bio-Rad, Hercules, CA, USA) and then were transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). Membranes were blocked with 1% BSA (Sigma Aldrich Inc., St. Louis, MO, USA) before incubation with Mu-Sda1 immunized mouse sera (3 × 10³-fold dilution). Bound antibody was detected by using goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP)-conjugate (Southern Biotech, Birmingham, AL, USA). The antibody-recognized band was visualized by ECL Western Blotting Substrate (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). For detection of cross-reaction of anti-Sda1 with recombinant DNase B and Homo sapien DNase I (Sino Biological, Beijing, China), the protein samples were separated by a 12% Tris·HCl SDS/PAGE gel, Western blotting assays were performed with mouse anti-Mu-Sda1 serum.