Precision plus protein standards all blue

The protein composition was tested using polyacrylamide gel electrophoresis methods—native PAGE and SDS-PAGE [47 (link)]. The total ESP and HSP fractions and the composition of thermostable protein were analyzed using PAGE and polypeptides—SDS-PAGE.
The concentration of the starting gel was 4%, the separating gel was 10%. The protein amount that was placed in one track was 50 µg. The distribution of the proteins using both PAGE and SDS-PAGE was carried out using ProteanII xi Cell (Bio-Rad, Hercules, CA, USA) and Multigel-Long (Biometra, Göttingen, Germany) apparatus, 10 mA for the starting gel, and 25 mA for the separating gel. Gels were visualized using Coomassie brilliant blue R250.
Molecular masses were estimated according to the localization in electrophoresis gels using standards: for non-denaturing electrophoresis, we used a Molecular Weight Marker Kit, 14,000–500,000 range (Sigma, JAV, St. Louis, MA, USA); for denaturing electrophoresis, we used Precision plus Protein Standards All Blue, 10,000–250,000 range (Bio-Rad, JAV).

Precast TGX 4–15% Midi Gel (Bio-Rad Laboratories, Hercules, CA, USA) were used in a Tris-Glycine buffer system. The protocol was adapted from previous studies [26 (link),27 (link)]. Briefly, samples were mixed with 1 × 1 Laemili buffer and 1% v/v of 1.4 M DTT. Each sample was incubated at 95 °C for 20 min. Precision Plus Protein™ Standards All Blue (Bio-Rad) was used as a protein marker. Samples were diluted 1:4 with distilled water before its addition to the gel. The gel was run at 200 V (400 mA). Coomassie staining solution was used to stain the total protein in the SDS-PAGE. For the western blot analysis, proteins were blotted using Trans-Blot^® turbo system (Bio-Rad) with 0.2 μm nitrocellulose membranes and blocked with 5% (w:v) skimmed milk overnight at 4 °C. Detection of Gag-eGFP protein was performed by incubation with primary mouse monoclonal antibody against HIV-1 p24 (ab9071, Abcam, Cambridge, UK), diluted 1:1000 in PBS-T for 2 h. Anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Abcam), diluted 1:1000 in PBS-T was used as a secondary antibody. Finally, the membrane was incubated with enhanced chemiluminescence (ECL) reagent (Bio-Rad) and visualized using a ChemiDoc imager (Bio-Rad).

All commercial chemicals were of the highest available grade. All powdered reactants and solutions for electrophoresis were from Sigma Chemical Co. (Milano, Italy). All the stock solutions for cell culture were from Euroclone (Celbio Milano, Italy). Precision Plus Protein Standards (All Blue) were from Bio-Rad (Milano, Italy) (Cat# 1610373). Complete protease inhibitor cocktail was from Roche Diagnostics S.p.A (Milano, Italy) (Cat# 11836145001). All the reagents used for histopathological analyses were from Bio-Optica (Milano, Italy).