Ni nta magnetic beads

To further assess protein binding, Ni-NTA Magnetic Beads (ThermoFisher # 88831) were used to compete with pre-bound proteins on the liposomes with a mass ratio of [CSP: Pfs230D1+ : CoPoP] = [1:0.5:1, 2, 3 or 4]. Sufficient beads were added to ensure full binding of the free proteins in the sample. The samples were incubated with the beads for 30 min before the supernatant and magnetic beads were separated and collected using a magnetic separator (ThermoFisher # 12321D). The beads were then resuspended in PBS. Denaturing reducing loading dye was then added to all samples (supernatant and beads) and heated to 100 °C for 10 min. The samples were then loaded into Novex 4–12% Bis-Tris acrylamide gel (Invitrogen # NP0321BOX) and subjected to PAGE and bands were visualized with Coomassie staining.

For co-purification of TatC_HIS, TatB and SufI_FLAG, cultures of DADE-P carrying either pFAT75ΔA-sufI_FLAG (control plasmid) or pBC_HIS-sufI_FLAG derivatives were grown as for membrane fraction preparation, but in the presence of 1 mM IPTG. After sonication, clarified cell lysates were solubilized overnight with 1.5 % digitonin in Buffer 1 (20 mM Tris-HCl, pH7.5, 200 mM NaCl, 50 mM imidazole, 10 % glycerol) and the soluble fraction separated by ultracentrifugation. TatC_HIS complexes with TatB and SufI_FLAG were captured with Ni-NTA magnetic beads (ThermoFisher), which were washed three times with wash buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 25 mM imidazole, 0.5 % digitonin) before elution with 1× SDS-PAGE loading buffer (Brand) by mild thermal treatment (53 °C for 10 min). Samples were then run on SDS-PAGE gradient gels (BioRad) and analysed by Western blotting with anti-TatB [15 (link)], anti-His (Invitrogen) or anti-FLAG (Sigma) antibodies. Blots were developed with ECL (BioRad).

To further check protein binding stability, Ni–NTA Magnetic Beads (ThermoFisher # 88831) were used to compete with pre-bound proteins to the liposomes (1:4 mass ratio of total protein: CoPoP). Sufficient beads were added to ensure full binding of the free proteins in the sample. The samples were incubated with the beads for 30 min before the supernatant and magnetic beads were separated and collected using a magnetic separator (ThermoFisher # 12321D). The beads were the resuspended in PBS. Denaturing reducing loading dye was then added to all samples (supernatant and beads) and heated near 100 °C for 10 min. The samples were then loaded into Novex 4–12% Bis–Tris acrylamide gel (Invitrogen # NP0321BOX) and subjected to PAGE and bands were visualized with Coomassie staining.

Experiments were performed with purified His₆-RpoA and extract of the E. coli BL21 pTOPO-SicA-FLAG strain. For this 100 μL of the cell-free extract were mixed with 50 μL of purified His₆-RpoA, 50 μL of Ni–NTA magnetic beads (Thermo Scientific) and 1X interaction buffer and let to interact for 2 h in agitation at ~ 4 °C. Samples were collected with a magnetic stand (New England Biolabs) for 2 min and washed four times with low imidazole buffer. After the last washing step supernatant was removed carefully, 30 μL of Laemmli buffer were added to the beads and samples were boiled for 10 min. Samples were resolved in a 12% SDS-PAGE, Western blot was performed by transferring the proteins to a PVDF membrane and using anti-FLAG-HRP antibodies (1:5,000) (Abcam AB49763) and HisProbe-HRP (1:5,000) as suggested by the manufacturers. Membrane development was done as described above.

To assess RBD or Spike protein binding stability in particle form after lyophilization and reconstitution, a Ni-NTA competition assay was carried out as recently described (17 (link)). Ni-NTA magnetic beads (Thermo Fisher Scientific, catalog no. 88831) were added to the liposome-incubated antigens (1:4 mass ratio of total protein:CoPoP) or free antigens in PBS. Following incubation with the beads for 30 min at RT, the supernatant and beads were separated and collected using a magnetic separator (Thermo Fisher Scientific, catalog no. 12321D). Denaturing reducing loading dye was then added to the samples (supernatant and beads) and heated at 95°C for 10 min. The samples were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) using Novex 4 to 12% bis-tris acrylamide gels (Invitrogen, catalog no. NP0321BOX).

Bacterially expressed ZIKV EDIII proteins (C-terminal 6 × His-tag) were conjugated to Ni-NTA Magnetic beads (Thermo Scientific) following manufacturers protocol. In short, 1 mg of Ni-NTA beads were washed in equilibration buffer and incubated on a rotator with 60 µg of ZIKV-EDIII or MBP-His (control depletions) for 1 h at 37 °C. Next, the beads were washed in equilibration buffer and divided in two tubes for two rounds of depletions. Mouse immune serum was diluted 1:20 in PBS and incubated with magnetic beads for 1 h at 37 °C on a rotator. Following incubation, the depleted serum was separated from the beads and stored at 4 °C for subsequent IgG ELISA and neutralization assays. The percentage of ZIKV EDIII-binding antibodies was calculated by dividing the EDIII-depleted IgG titers by the control-depleted IgG titers. The percentage of ZIKV EDIII neutralizing antibodies was calculated by dividing the EDIII-depleted neut₅₀ by the control-depleted neut₅₀.

The HEK cells were co-transfected with N-terminally His-tagged Human mTOR Gene Copoeia Cat# EX-Mm31144-M48) and C-terminally Avi-tagged Human RAPTOR (Gene Copoeia Cat# EX-E0449-M62). The IP was performed with Ni-NTA magnetic beads (Thermo Fisher, Cat# 88831). Westerns were developed using IRDye^® 680RD Streptavidin (LiCor Biosciences, Cat# 926-68079) and anti-mTOR antibody, before the protein isolation cells were pre-treated with a 10 µM concentration of indicated drugs for 2 h. The bands intensities were quantified using Image Studio Software (Licor Biosciences Lincoln, Nebraska).