Precision plus protein all blue prestained standard

Samples of cell culture supernatant or purified N-CD protein, previously precipitated with TCA and sodium deoxicolate or not, were analyzed by SDS-PAGE as described by Laemmli [18 (link)]. SARS-CoV-2 N protein (2–419 aa) expressed in Escherichia coli (CIGB-Sancti Spiritus, Cuba) was used as positive control and 1–2 μg of protein were applied per gel. Samples and the positive control were loaded in 12.5% SDS-PAGE. After running, gels were used for immunoblotting analysis or stained with Coomassie Brilliant Blue G-250. Precision Plus Protein All Blue Prestained Standard (Bio-Rad, Hercules, USA) or Broad Range Protein Molecular Marker (Promega, Madison, USA) were used as protein standards (10–250 kDa).

All samples were denatured in loading buffer (4x, 0.25 M Tris/HCl pH 6.8, 8% [w/v] SDS, 20% [w/v] glycerol, 0.04% [w/v] bromophenol blue, 0.4% [v/v] β-mercaptoethanol) for 10 min at 90 °C. The samples, Precision Plus Protein™ All Blue Prestained standard (Bio-Rad Laboratories, Hercules, USA), a 12.5% (w/v) polyacrylamide gel, and the appropriate buffer (0.025 M Tris, 0.192 M glycerol, 1% [w/v] SDS) were then used for SDS-PAGE according to Laemmli [45 (link)]. After separation, the proteins were either used for Coomassie staining (45% [v/v] methanol, 10% [v/v] acetic acid, 0.25% [w/v] CuSO₄, 0.2% [w/v] Coomassie brilliant blue G250), or they were transferred to a nitrocellulose membrane (GE Healthcare Europe GmbH, Freiburg, Germany) using a semi-dry transfer procedure [46 (link)]. The membrane was incubated with 5% milk powder in TBS (10 mM Tris/HCl and 150 mM NaCl, pH 7.5) for 1 h. A Strep-Tactin horseradish peroxidase conjugate (IBA, Göttingen, Germany) was used according to the manufacturer’s instructions to detect all Strep-tag II fusion proteins.