Anti his tag antibody

All culture samples were centrifuged and cell pellets rinsed three times with PBS buffer and stored at −80°C for subsequent Western blot and/or culture GFP fluorescence analysis. Each cell pellet sample was thawed, resuspended in PBS buffer, and diluted to OD₆₀₀ = 0.3 to measure the culture GFP fluorescence with a Hitachi F-2500 fluorescence spectrophotometer. To prepare protein extracts for Western blot analysis, cell pellets were resuspended in 10% TCA buffer, followed by homogenization using Mini-Beadbeater-16 (Biospec model 607, Bartlesville, OK) with zirconia/silica beads (0.5 mm) in 3 × 1 min bursts. The extracted protein pellet was resuspended in a resuspension buffer as described previously (Cox et al., 1997 (link)). The protein concentration in the extract was measured using the Nanodrop ND-1000 spectrophotometer. The protein extract is then subject to SDS-PAGE and Western blot analysis. SDS-PAGE was performed using the 12% polyacrylamide gel; about 5 µg of total soluble protein for each sample was mixed with 5× loading buffer and subjected to electrophoresis. Proteins separated in SDS-PAGE gel were electroblotted onto a polyvinylidene difluoride (PVDF) membrane, and probed with anti-GFP antibody or anti-His Tag antibody (Genscript, Piscataway NJ), as described previously (Zhang et al., 2017 (link)).

Proteins were separated by SDS-PAGE and transferred to a PVDF membrane by dry transfer using the iBlot dry blotting system from Invitrogen (Waltham, MA, USA). Membranes were blocked for 1 h at room temperature in 5% w/v milk or BSA in TBS-T (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 0.05% Tween), followed by incubation with primary antibody overnight at 4 °C. Primary antibodies: anti-His-tag antibody (#A00186, GenScript, 1:5000) (Piscataway, NJ, USA), anti-His-tag antibody (#12698, Cell Signaling, 1:1000) (Danvers, MA, USA), anti-NEIL2 antibody (#124106, Abcam, 1:500) (Cambridge, UK), anti-P35 antibody (anti-p35, SantaCruz, #sc-518009, 1:2500) (Dallas, TX, USA), and anti-actin antibody (#A2228, Sigma, 1:10,000) (Merck, Darmstadt, Germany). Subsequently, the membrane was washed in TBS-T and incubated with secondary anti-mouse IgG Horseradish Peroxidase Linked (GE Healthcare, #NA931, 1:5000) (Chicago, IL, USA) or anti-rabbit IgG Horseradish Peroxidase Linked (GE Healthcare, #NA934, 1:5000) (Chicago, IL, USA) antibody for 1 h at room temperature, followed by additional washing in TBS-T and detection with ECL Prime Western Blotting Detection Reagent from GE Healthcare (Chicago, IL, USA) according to the manufacturer’s protocol.

Nanostructured proteins were produced in E. coli following the method described in Torrealba et al. (9 (link)) and Thwaite et al. (12 (link)). In short, E. coli transformed with the plasmid of interest was cultured in LB with the appropriate antibiotic and recombinant protein expression was induced at OD_550nm 0.5–0.8 with 1 mM IPTG (Panreac, Barcelona, Spain). IBs were isolated after 3 h additional incubation at 37°C via enzymatic and mechanical disruption of the cells according to Torrealba et al. (10 (link)), followed by sterility monitoring (12 (link)). Purified nanoparticles, named here IB^{frg16G−VHSV}, IB^TNFα and IB^iRFP [an inclusion body made of a non-immunogenic phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm (13 (link))], were stored at −80°C until use. Quantification was performed by western blot using an anti-His-tag antibody (Genscript, Piscataway, NJ, USA) and calculating the protein concentration from a standard curve using Quantity One software (Biorad, Hercules, CA, the USA). For flow cytometry or confocal microscopy, IB^{frg16G−VHSV} and IB^TNFα were conjugated with fluorescent Atto-488 NHS ester (Sigma-Aldrich) following manufacturer's instructions.

RBD proteins described above (Supplemental Table 3) were covalently coupled onto magnetic multiplex beads as previously described (65 (link)). Briefly, each respective RBD protein was coupled to a distinct magnetic carboxylated bead region (Bio-Rad), using a 2-step carbodiimide reaction at a ratio of 1 million beads to 10 μg of each RBD protein. Recombinant influenza hemagglutinin from H1N1 (A/Cali/07/2009) (11085-V08H, Sino Biological) and SARS-CoV-2 Spike S1 (40591-V08H, Sino Biological) were also coupled using identical protocols as control proteins. Coupling efficiency of the recombinant RBD was assessed using an anti-His Tag antibody (A00174, GenScript Corporation). A complete list of the magnetic bead regions and respective RBD variants is in Supplemental Table 3.