Protein samples were resolved by SDS-PAGE using 12 or 15 % gels for 80 min at 100 V (Mini-Protean Cell; Amersham Biosciences, Piscataway, NJ, USA) and then electro transferred (120 V for 2 h) onto nitrocellulose membranes (Hybond ECL; GE Healthcare, Little Chalfont, UK). Membranes were blocked and then incubated with the appropriate primary antibody, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:3,000) in PBS-Tween-20. After further washing with PBS-Tween-20, the membranes were developed with western lightning enhanced chemiluminescence reagent (Pearce, Rockford, IL, USA). The membranes were stripped if necessary. All the antibodies used in this work are presented in Table 1 .
Mini protean cell
Manufactured by Cytiva
Sourced in United States
The Mini-Protean Cell is a compact and versatile electrophoresis system designed for protein separation and analysis. It is capable of performing SDS-PAGE, native PAGE, and other electrophoretic techniques. The system is compact and easy to set up, making it suitable for a variety of laboratory applications.
Automatically generated - may contain errors
4 protocols using mini protean cell
1
Western Blot Protein Analysis
2
Western Blot Protein Detection Protocol
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Supernatant samples were resolved by SDS-PAGE using 12% or 15% gels for 80 min at 100 V (Mini-Protean Cell; Amersham Biosciences, Piscataway, NJ, USA), and further electrotransferred (120 V for 2 h) onto PDV (Hybond ECL; GE Healthcare, Little Chalfont, UK). Membranes were blocked with semi-skimmed Milk/PBS-Tween-20 and then incubated with the appropriate primary antibody such as anti-His, pre-pandemic serum samples, or monoclonal anti-S1, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) in PBS-Tween-20. After further washing with PBS-Tween-20, the membranes were developed with western lightning-enhanced chemiluminescence reagent (Pearce, Rockford, IL, USA).
3
Western Blot Protein Analysis
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Each 50-µL fraction was mixed with 10 µL of Laemmli loading buffer and then heated at 95°C for 10 min. Sample proteins were resolved by SDS-PAGE using 12% Tris-HCl gels for 80 min at 160 V (Mini-Protean Cell; Amersham Biosciences, Piscataway, NJ, USA) and then electrotransferred (120 V for 2 h) onto nitrocellulose membranes (Hybond ECL; GE Healthcare, Little Chalfont, UK). Air-dried membranes were blocked and then incubated with the appropriate primary antibody, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1∶3000) in PBS plus Tween-20. The membranes were stripped if necessary. After further washing with PBS plus Tween-20, the membranes were treated with Western Lightning Enhanced Chemiluminescence Reagent (Pearce, Rockford, IL, USA), and immunoreactive proteins were detected by exposure to film (Kodak, Rochester, NY, USA).
4
Zika Virus Infection Kinetics in Vero Cells
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Vero cells at 80% confluency were infected with ZIKV at an MOI of 5. Infection kinetics at 8, 12 and 24 h were performed. Uninfected Vero cells and Vero cells treated with a heat-inactivated virus (mock) were used as controls. Cells were then lysed with RIPA buffer (100 mM Tris-HCl pH 8.3, 2% Triton X-100, 150 mM NaCl, 0.6 M KCl, 5 mM EDTA) in the presence of one tablet of the protease inhibitor cocktail per 25 mL (Complete, Invitrogen). Cell lysates were analyzed by 12% SDS-PAGE for 80 min at 100 V (Mini-Protean Cell, Amersham Biosciences, Piscataway, NJ, USA) and transferred into nitrocellulose membranes (BIO-RAD) was carried out at 120 V for approximately 2 h). Membranes were blocked with PBS-Tween-5 % milk for 1 h and then washed four times with PBS-Tween. Membranes were then incubated overnight with the primary antibodie (lab-made for NTDyn (Additional file 1 ), and anti-E mAb 4G2 (Merk-Millipore)) at 4 °C. After this incubation, the membranes were washed again with 1X PBS and incubated with HRP-coupled secondary (Invitrogen) for 1 h. After another round of 4 washes with PBS-Tween, the membranes were developed in the presence of the chemiluminescence developer reagent (Thermo Scientific) following the manufacturer’s instructions, using the ChemiDoc kit (BIO-RAD) and digitalized with the ChemiDoc XRS System (BIO-RAD).
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