Anti rabbit igg horseradish peroxidase hrp linked antibodies

Cell were lysed at 4 °C for 30 min with lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% [v/v] Triton X-100; protease inhibitor cocktail [Roche]). Cell lysates were centrifuged at 12,000 × g for 10 min and clarified supernatants were collected. The total protein concentrations were determined by the Bradford method against a bovine serum albumin standard. Protein was boiled in SDS-PAGE sample buffer (125 mM Tris–HCl, pH 6.8; 4% [v/v] SDS; 20% [v/v] glycerol; 0.004% [w/v] bromphenol blue), and separated by 10% SDS-PAGE. Gels were electro-blotted onto a polyvinylidene fluoride membrane (Pall) using a semi-dry transfer system (Bio-Rad). DENV-2 E and CA proteins were detected with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV C polyclonal antibody (Novusbio), respectively. DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS). Endogenous β-tubulin was detected with a mouse anti-β-tubulin monoclonal antibody (Sigma Aldrich). Primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies or anti-mouse IgG HRP-linked antibodies (Cell Signaling Technology).

Culture supernatants from HEK-DI-290-ORF cells or DENV-2 infected HEK 293T cells, or purified DENV DIP supernatant, were ultracentrifuged at 100,000 × g for 1 h at 4°C. The pelleted materials were boiled in SDS-PAGE sample buffer (125 mM Tris-HCl [pH 6.8], 4% [vol/vol] SDS, 20% [vol/vol] glycerol, 0.004% [wt/vol] bromophenol blue) and separated by 4 to 20% precast SDS-PAGE gels (Bio-Rad). Gels were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Pall) using a semi-dry transfer system (Bio-Rad). DENV-2 E and CA proteins were detected with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV CA polyclonal antibody (Novus Biologicals), respectively. Primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies (Cell Signaling Technology).

Culture supernatants from HEK-DI-290-ORF cells or DENV-2 infected HEK 293T cells, or purified DENV DIP supernatant, were ultracentrifuged at 100,000 × g for 1 h at 4°C. The pelleted materials were boiled in SDS-PAGE sample buffer (125 mM Tris-HCl [pH 6.8], 4% [vol/vol] SDS, 20% [vol/vol] glycerol, 0.004% [wt/vol] bromophenol blue) and separated by 4 to 20% precast SDS-PAGE gels (Bio-Rad). Gels were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Pall) using a semi-dry transfer system (Bio-Rad). DENV-2 E and CA proteins were detected with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV CA polyclonal antibody (Novus Biologicals), respectively. Primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies (Cell Signaling Technology).