Anti human igg h l hrp

The EphA2-Fc and EphA4-Fc bound to the transfected cells was determined by cell enzyme-linked immunosorbent assay (CELISA). CHO-K1 cells were transiently transfected with control plasmid pcDNA 3.1, EBV gH/gL, and KSHV gH/gL. Soluble EphA2-Fc and EphA4-Fc were prepared by transfecting the CHO-K1 cells with EphA2-Fc and EphA4-Fc constructs and used to overlay epithelial cells (5 × 10⁴ cells/well) in 96-well plates in triplicate. After incubation for 2 h at 4°C, the cells were incubated with anti-human IgG (H&L) (HRP) (ab6759, 1:1,000; Abcam) against the Fc region for 30 min and fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS for 15 min, followed by three PBS washes. 3,3′,5,5′,-Tetramethylbenzidine (TMB) one-component HRP microwell substrate was added, and the amounts of bound EphA2-Fc and EphA4-Fc were determined by measuring the absorbance at 380 nm with a PerkinElmer Victor plate reader. Binding activity was standardized in comparison to EphA2-Fc binding to EBV gB, which was set to 100%.

Expi293F culture supernatants were collected 3 days after transfection, harvested via spinning at 7,000 × g for 15 minutes, and filtered through a 0.22-μm filter. Samples were diluted in SDS-PAGE Laemmli loading buffer (Bio-Rad), boiled at 95 °C, and run on a 4–20% Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system. Blots were blocked in 5% milk / PBST (1X PBS [pH 7.4], 0.1% Tween 20) and then washed with PBST. In-house made primary antibodies CR3022, COVA2–15, and CB6 (approximate concentrations 0.8 – 1.3 mg/mL) were added at a 1:10,000 dilution in PBST. Blots were washed with PBST and secondary rabbit anti-human IgG H&L HRP (abcam ab6759) was added at 1:10,000 in PBST. Blots were developed using Pierce ECL substrate and imaged using a GE Amersham Imager 600.

The purified HIV-1 samples (200 ng) were loaded into the wells of the SDS-PAGE gel and allowed to run at 110 V for 45 min. After SDS electrophoresis, the protein bands after SDS electrophoresis were transferred to the nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The nitrocellulose membrane was blocked in 5% BSA (containing 0.1% Tween 20) for 2 h on a shaker. The 5% BSA was discarded, and a 1:1000 dilution of the primary antibody (Anti-HIV protease, EXBIO Praha, Vestec, Czech Republic) was added to the nitrocellulose membrane, left on the shaker for 1 h, and then stored overnight at 4 °C. The nitrocellulose membrane was then subsequently washed 5 times in wash buffer (10× Tris-Buffered Saline (TBS), and a 1:1000 dilution of the secondary antibody (Anti-Human IgG H&L, HRP, Abcam, United Kingdom)) was added and placed on the shaker for 2 h. The nitrocellulose membrane was then washed a second time. The Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, MA, USA) was added to the membrane and visualized in a light-sensitive film.