Horseradish peroxidase hrp conjugated goat anti mouse antibody

The proteins in an SDS-polyacrylamide gel were transferred onto an Immobilon^®-P PVDF transfer membrane (Millipore Sigma) by electroblotting at 250mA for two hours in ice cold transfer buffer (25mM Tris, 192mM glycine and 10% methanol). The blots were blocked at room temperature for one hour in Blotto (PBT: 10% PBS and 0.1% Tween-20, and 3% skim milk). Anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) at a dilution of 60,000-fold in Blotto was incubated with the blot for one hour at room temperature. After washes with PBT, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (ThermoFisher Sci.) at a dilution of 3,000-fold in Blotto was added and incubated for one hour at room temperature. After washes with PBT, HRP was detected using SuperSignal^™ West Femto Maximum Sensitivity Chemiluminescent Substrate (ThermoFisher Sci.). Digital images were recorded using a ChemiDoc^™ Imaging System (Bio-Rad). For some experiments, the membrane was stripped for one hour at room temperature using Restore^™ Western Blot Stripping Buffer (ThermoFisher Sci.) to remove the anti-FLAG antibody and was blocked at room temperature for one hour in Blotto followed by incubation with anti-β-tubulin monoclonal antibody (E7 concentrated from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) at a dilution of 1,500-fold in PBT.

These experiments were performed as described previously [18 (link)]. For SDS-PAGE, cell culture supernatants or purified proteins were boiled for 10 min with loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 1% DTT), and the samples were separated by 10–12% SDS-PAGE. For BN-PAGE, purified protein samples were prepared in 2 × Protein Native PAGE Loading Buffer (TaKaRa, Dalian, China) and loaded onto a 4 to 12% Bis-Tris NuPAGE gel (Invitrogen, Carlsbad, CA, USA). Western blot analysis was performed as described previously [18 (link)]. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 µm; Millipore, Cork, Ireland). PVDF membrane was blocked with 5% nonfat milk and subsequently incubated with an anti-DC-SIGN MAb (Santa Cruz, clone 120507, Dallas, TX, USA) for 1 h at room temperature. After 3 washes with TBS-Tween (200 mM NaCl, 50 mM Tris-HCl, 0.1% Tween-20), the membrane was incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Protein bands were visualized following incubation with enhanced chemiluminescence (ECL) (Millipore, Billerica, MA, USA).

For detection of CCHFV L polymerase (448kDa), lysate samples from tc-VLP donor cells were run through Criterion XT Tris-acetate precast gels (3–8% gradient) (Biorad) in a XT tricine buffer (Biorad), for 1 h 40 min at 150V. Proteins were transferred on an EtOH-activated polyvinylidene fluoride (PVDF) membrane (Millipore) by wet blotting over-night at 40 mA and 4°C, using Tris-glycine transfer buffer (5.8 g/L Tris (Acros), 2 g/L glycine (Roth), 10% absolute EtOH (Roth)). The CCHFV L was detected via its V5-tag (1:5000) (Novex, Life-technologies) and a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Thermo Fisher) (1:20,000). Quantification of the signals was performed using the Image Lab 4.0 software.
For detection of CCHFV Gn and N in donor cell supernatants, tc-VLPs (and CCHFV strain IbAr10200 as control) were produced, concentrated by ultracentrifugation through a 20% sucrose cushion, and their protein content analyzed by Western blotting (10% SDS-PAGE) and chemiluminescence quantification as described in [32 (link)].
All quantification values are shown as a percentage of the signal in wt L donor cells.