Coomassie brilliant blue r 250

Cloning of NcGRA6 gene and expression of recombinant proteins of N. caninum dense granule protein 6 fused with glutathione-S-transferase (NcGRA6 + GST) and glutathione-S-transferase (GST) were performed as previously described [21 (link)]. In brief, the PCR products digested with EcoRI and XhoI were inserted into the pGEX-4T1 plasmid vector (Amersham Pharmacia Biotech, Madison, CA, USA). Recombinant NcGRA6 was expressed as glutathione S-transferase (GST) fusion protein (NcGRA6+GST) in Escherichia coli BL21 (DE3) (New England BioLabs Inc., Ipswich, MA, USA). The purity and amount of the proteins were determined by running sample on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 (MP Biomedicals Inc., Illkirch-Graffenstaden, France). The proteins were dialyzed in PBS and filtered with a 0.45-μm low-protein binding Supor membrane, and the endotoxin was removed using Acrodisc Units with Mustang E Membrane (Pall Life Sciences, Ann Arbor, MI, USA) for subsequent use for in vivo experiments. In addition, level of endotoxin was measured with limulus amebocyte lysate reagents (Seikagaku Inc., Tokyo, Japan), and no endotoxin was found in the tested protein lots.

Approximately equal amounts of each recombinant protein and bovine serum albumin (as a negative control in western blotting) were mixed with 5 × loading buffer, boiled for 10 min, and then loaded onto 12% SDS-PAGE. The gel was stained with Coomassie brilliant blue R250 (MP Biomedicals, Santa Ana, USA) or electrophoretically transferred to polyvinylidene difluoride membranes (Milipore, San Diego, CA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated with anti-histidine monoclonal antibody (1:5000 dilution, Abcam, Cambridge, UK) or anti-ASFV swine serum (1:300 dilution, Diagnostic Products Center, LVRI, China) overnight at 4 °C. After washing five times with PBS containing 0.05% Tween-20 (PBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:5000 dilution, Abcam) or goat anti-pig antibody (1:5000 dilution, Abcam) for 1 h at room temperature. After being washed with PBST, the membranes were developed using enhanced chemiluminescence reagent (Thermo Fisher Scientific).