Horseradish peroxidase hrp conjugated goat anti mouse and goat anti rabbit antibodies

Vector supernatants were concentrated by ultracentrifugation (100-fold), electrophoresed on 4 to 12% Bis-Tris protein gels (Bio-Rad), and transferred to polyvinylidene difluoride membranes using a Trans-Blot turbo transfer system (Bio-Rad). Membranes were blocked with 5% milk in PBST buffer (PBS plus 0.1% Tween 20). The S1 subunit of Spike was detected using SARS-CoV-2 (COVID-19) Spike S1 antibody at 1:1,000 (9083; Prosci); the S2 subunit was detected using anti-SARS-CoV/SARS-CoV-2 (COVID-19) spike antibody clone [1A9] at 1:1,000 (GTX632604; GeneTex); and HIV-1 p24 was detected using a polyclonal anti-HIV-1 SF2 p24 rabbit antiserum at 1:6,000 (obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH; ARP-4250, contributed by DAIDS/NIAID; produced by BioMolecular Technologies). VSV M protein was detected using anti-VSV M antibody clone (23H12) (EB0011; KeraFast, Boston, MA) at 1:1,000. Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit antibodies were used as secondary antibodies (Santa Cruz Biotechnology, Dallas, TX). Blots were imaged by Amersham ECL Prime Western blotting detection reagent (GE Healthcare, Chicago, IL) and Chemidoc (Bio-Rad). Densitometry was measured using ImageJ software (http://rsb.info.nih.gov/ij/).