Alkali soluble casein

Western blot analysis was performed as described in Ogrina et al. (2022) (link). First, samples for EMV-Feld1-containing VLPs were loaded and run on 17-well 1.0 mm Bolt 4–12% Bis-Tris Plus gel (Thermo Fisher Scientific, USA). The corresponding proteins were then transferred onto an Amersham Protran 0.45 μm nitrocellulose membrane (GE Healthcare, Piscataway, USA) for antibody detection using a semidry blotting apparatus (45 mA, 45 min). A membrane was then blocked with 1% alkali-soluble casein (Merck-Millipore, USA) for 1 h at RT followed by incubation overnight at 4°C in anti-EMV and anti-Feld1 polyclonal antibodies (Ab’s) (dilution 1: 1,000 in PBS with 1% alkali-soluble casein, in-house produced). After a 15 min washing step with TBS buffer (150 mM NaCl; 10 mM Tris pH 7.5), membranes were incubated for 3 h with horseradish peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich, USA; 1:1,000 in PBS with 1% alkali-soluble casein) at RT. After a repeated washing step with TBS buffer, signals were detected by incubating the membrane in TBS buffer supplemented with peroxidase substrates (0.002% o – dianisidine and 0.03% hydrogen peroxide).

Cy5™-streptavidin (Cy5-SA) was purchased from GE Healthcare (Little Chalfont, UK). Freshly deprotected arrays were used in each experiment. Streptavidin binding to all arrays was performed with 0.5 µg/ml Cy5-SA either in binding buffer containing 10 mM Tris-HCl (pH 7.4), 1% alkali-soluble casein (EMD Millipore), 0.05% Tween-20 or in 10 mM Tris-HCl (pH 7.4), 4% BSA (Roche, Basel, Switzerland), 0.05% Tween-20 in a 30 mL PAP Jar container (Evergreen Scientific, Vernon, CA) overnight at 4° C. After incubation, arrays were washed in 20 mM Tris-HCl (pH 7.8), 0.2 M NaCl, 1% SDS or 1X TBS (pH 7.4) for 30 sec followed by a 1 min wash in water, and then dried by spinning in a microcentrifuge equipped with an array holder.

Extracted membrane proteins from spheroplasts or IMV were separated on 16% Tricine-SDS-PAGE. Proteins were then transferred to PVDF (Sigma-Aldrich, Millipore) in a semi-dry blot procedure (overnight, 30 mA). The membrane was blocked with 1% Alkali-Soluble Casein (Sigma-Aldrich, Novagen) in TBS buffer (10 mM Tris-Cl; pH 7.5; 150 mM NaCl) for 1 h and washed twice in TBS. Then the membrane was incubated with primary anti-His-tag antibody (1:1000 in blocking solution, 60 min) and washed with TBSTT buffer (20 mM Tris-Cl; pH 7.5; 500 mM NaCl; 0.2% Triton X-100; 0.05% Tween 20) and TBS buffer. Then, the membrane was incubated with secondary goat-anti-mouse-IgG coupled to alkaline phosphatase (1:5000 in blocking solution, 60 min). After extensive washing with TBSTT buffer, the alkaline phosphatase was detected with chromogenic substrate BCIP/NBT according to the manufacturer’s protocol (Sigma-Aldrich).