Pierce bradford protein assay kit

Recombinant CNA expressed in Escherichia coli was used for the current experiment. Briefly, the full-length coding sequence of the CNA (Appendix 1) gene was cloned into the pET-30a expression vector with an NH2-terminal polyhistidine epitope tag. Primers contained NdeI and HindIII restriction enzyme sites (645 amino acids with molecular weight = 72,480.5). Recombinant CNA/pET-30a was transformed into E. coli strain BL21 Star (DE3) cells (Life Technologies, Grand Island, NY). A single colony was inoculated with 1.0 mM isopropyl beta-D-thiogalactopyranoside (Amresco, Cleveland, OH). The recombinant CNA was purified using Ni-NTA His Bind Resin column (NOVAGEN, Darmstadt, Germany) and the concentration was determined by Bradford protein assay (Pierce Bradford Protein Assay Kit, Thermo Scientific, Rockford, IL). The molecular weight of CNA was confirmed using Coomassie bright blue stained SDS-acrylamide gels.

Whole-cell lysates were prepared in RIPA lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate) supplemented with 1 mM PMSF, 1% SDS, and phosphatase inhibitors (1x PhosphoSTOP Phosphatase Inhibitor Cocktail, Roche Diagnostics, Cat#4906845001). Samples were lysed on ice for 20 min, sheared by passing cells through a G25 needle (B. Braun, Hypodermic Needle-Pro®, Cat#4658304), cleared by centrifugation (14,000 × g, 15 min), and quantified using Pierce™ Bradford protein assay kit (Thermo Scientific, Cat#23200). Lysates were boiled 5 min at 95 °C with 6x reducing Laemmli buffer (0.12 M Tris pH 6.8, 47% glycerol, 12% SDS, 0.6 M DTT, 0.06% bromophenol blue). Samples were separated on a polyacrylamide gel and transferred to PVDF membranes using the mini wet/tank blotting system (Bio-Rad). After blocking with 5% BSA in Tween-20/PBS, membranes were probed with primary antibodies prepared in blocking solution overnight at 4 °C on a roller, followed by incubation with horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature and ECL detection (Thermo Fisher, Cat#34096) by the ChemiDoc XRS + system (Bio-Rad). Primary and secondary antibodies used for immunoblotting are listed in Additional file 8: Table S1. Quantitative analysis of protein expression relative to GAPDH was done using Image Lab software (Bio-Rad).