Hrp labeled anti rabbit igg

Foreign and domestic commercial sources were used to acquire biological components and chemical reagents of analytical quality. DNA polymerase and T4 DNA ligase were acquired from TaKaRa Bio Inc. (Dalian, China), as well as the DNA marker, DNA restriction enzymes, E. coli DH10Bac cells, and Escherichia coli DH5 cells. Fetal bovine serum (FBS), GibcoTM Sf-900TM III SFM, and the pure LinkTM Quick Plasmid Miniprep Kit were all purchased from Thermo Fisher, USA). We used ExpiSf9TM cells that we originally purchased from Thermo Fisher, USA). Anti-His-Tag mouse monoclonal antibody and goat anti-mouse secondary antibody labeled with horseradish peroxidase (HRP) (Abcam, Cambridge, UK) Monoclonal rabbit antiDE-Loop BPV-VP2 (SanQ Biotechnology Co.Ltd., Beijing) and HRP-labeled anti-rabbit IgG (from Sigma-Aldrich;St.Louis, Missouri, USA) were used.

Cell lysates of iPSCs and iMSCs were prepared using RIPA lysis buffer (Beyotime, China) supplemented with 1 × phenylmethanesulfonyl fluoride (PMSF, 1 mM) and Protease Inhibitor Cocktail (Sigma-Aldrich). Protein in the samples was measured by a Pierce^TM BCA protein assay kit (Thermo Fisher Scientific, United States). Protein samples (15 μg) were loaded on an electrophoresis gel that was run for 2 h at 120 V, and the proteins were transferred to PVDF membranes (Millipore, United States). After blocking with 5% non-fat milk in 0.1% TBST (TBS containing 0.1% Tween-20), the membrane was incubated overnight with anti-LMAN1 antibodies (Abcam, England) at 4°C. The membrane was then incubated with HRP-labeled anti-rabbit IgG (Sigma-Aldrich) for 1 h at room temperature, and the signal was visualized using an ECL detection kit (Thermo Fisher Scientific). Then, the membrane was washed with stripping buffer and 0.1% TBST. After blocking, the membrane was incubated overnight with anti-β-actin antibodies (Sigma-Aldrich) at 4°C. The membrane was incubated with anti-mouse IgG (Sigma-Aldrich) for 1 h at room temperature, and the signal was visualized using an ECL detection kit (Thermo Fisher Scientific).

(i) By anti-ALP antibody. BmBBMVs proteins (10 µg/mL) coated on 96-well ELISA plates were pretreated with different concentrations of the anti-ALP or pre-immune antibodies (0.2–20 µg/mL) at 37°C for 1 h, then incubated with DpCPV virions (final amounts, 10 µg) at 37 °C for a further 1 h. The wells were washed with PBST and the bound virions were detected using a rabbit anti-DpCPV virion antibody followed by HRP-labeled anti-rabbit IgG (Sigma-Aldrich).
(ii) By BmALP proteins. DpCPV virions (final amounts, 10 µg) were pretreated with different concentrations of BmALP protein (0.02–2 µM) or BSA at 37 °C for 1 h, and then added to the BmBBMVs (10 µg/mL) coated in 96-well ELISA plates. After absorption for 1 h, the bound virions were detected as described above.