Xt mops buffer

Pooled leaf discs as described above were either homogenised directly in 100 µl Laemmli buffer or were macerated in the Rluc-PCA assay buffer and Laemmli buffer added. The samples were boiled for 5min and cooled on ice. Ten microliters of the homogenate were separated on a 12% 1-mm thick polyacrylamide gel (Criterion™ XT Bis-Tris precast polyacrylamide gel, Biorad, Hercules, CA, USA) in 1× XT-MOPS buffer (Biorad, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membrane and probed with primary and secondary antibodies. Antibodies were diluted in PBS-T 1% (w/v) skimmed milk powder as the following: rabbit α-HA (Sigma-Aldrich, St. Louis, MO, USA), 1:500; swine α-rabbit HRP-conjugate (Dako, Glostrup, Denmark), 1:1700; mouse α-FLAG M2 (Sigma-Aldrich), 1:1000; rabbit α-mouse HRP-conjugate (Dako, Glostrup, Denmark), 1:2000; mouse α-cMyc 9E10 (Sigma-Aldrich), 1:1000, where skimmed milk powder was omitted. Detection was performed with SuperSignal West Dura chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA).

Sample loading buffer (Bio-Rad XT sample buffer) and reducing agent (Bio-Rad) was added to the samples according to the manufacturer’s instruction and equivalent amounts of protein (50 µg as measured using the DC Protein Assay Kit (Bio-Rad) and proteins were separated on a 4 to 12% on a 12% gradient XT precast Criterion gel using XT-MOPS buffer (Bio-Rad) at 150–200 V. Subsequently, proteins were transferred onto a PVDF membrane. Membranes were blocked for 30 min in a 1:1 Tris-buffered saline (TBS)/Odyssey blocking solution (cat n° 927-40003, LI-COR, Lincoln, NE, USA) and probed by Western blotting. Following overnight incubation of primary antibody in TBS-T/Odyssey blocking buffer and three 10 min washes in TBS-T (0.1% Tween-20), membranes were incubated with secondary antibody for 30 min in TBS-T/Odyssey blocking buffer followed by 3 washes in TBS-T or TBS (last washing step). The following antibodies were used: mouse anti-GAPDH (Abcam, ab9484, 1:10,000), rabbit purified IgG anti-NAA10 (anti-hARD1, Biogenes GmBH [58 (link)], 1:1000) and anti-NAA15 (anti-NATH, Biogenes GmBH [58 (link)], 1:1000), anti-mouse (IRDye 800 CW goat anti-mouse antibody IgG, LI-COR, cat n°926-32210, 1:10,000) and anti-rabbit (IRDye 800 CW goat anti-rabbit IgG, LI-COR, cat n°926-3221, 1:10,000). The bands were visualized using an Odyssey infrared imaging system (LI-COR).

SDS-PAGE was used to compare the compositions of the dECMs. Protein extracts were prepared using the Pierce Protein Assay Kit according to manufacturer instructions and resuspended in 7.5 μL XT sample buffer/1.5 μL XT reducing agent (Bio-Rad), placed in a 95°C heating block for 10 min, and centrifuged for 2 min at 14,000 g. Samples were loaded into 1.0 mm 4–12% gradient Bis-Tris Criterion XT Precast Gel (Bio-Rad), with collagen I (Sigma Aldrich) and Precision Plus protein ladder (Bio-Rad) used as controls. Loaded gels were placed into XT MOPS buffer (Bio-Rad) and electrophoresed at 150 V for ~100 min. Gels were then stained with BioSafe Coomassie (Bio-Rad) for at least 2 h and washed with deionized water three times for 5 min each. Bands were visualized using a GE Image Scanner III, with images taken using ImageQuant TL software (GE Healthcare).

250 YU aliquots from N or N-PbCS cultures were lysed and clarified as described above and stored at −80 °C for western blot (WB) analysis of antigen concentration after each immunization. Stored samples contained 28 μl of N-PbCS lysate or of a 1/125 dilution of N lysate, 10 μl of XT Sample Buffer 4× (161-0791, Bio-Rad) and 2 μl of XT Reducing Agent 20× (161-0792, Bio-Rad). WB was performed as previously described [6 (link)] in denaturing conditions on 4–12% Bis–Tris polyacrylamide gels with XT MOPS buffer (Criterion 345-0123, Bio-Rad) using the Color Plus Prestained Protein Ladder (7-200 kDa; P7711 BioLabs). Anti-N monoclonal antibody (3E1, Abnova) was used at 1/2000 dilution to detect N-PbCS and N proteins in yeast lysate samples.

VLP and producer cell lysates were added sample loading buffer (XT sample buffer, Bio-Rad) and reducing agent (XT reducing agent, Bio-Rad) according to the manufacturer's instructions. Proteins were separated on a 4–12% gradient XT precast Criterion gel using XT-MOPS buffer (Bio-Rad) at 150 V and subsequently transferred onto a PVDF membrane. Membranes were blocked for 30 min in a 1:1 Tris-buffered saline (TBS)/Odyssey blocking solution (cat no. 927-40003, LI-COR) and probed using primary antibodies (1/1000 dilution, rabbit anti-PFN1, Abcam, cat no. 124904; 1/5000 dilution, mouse anti-Gag Abcam, cat no. 9071; 1/10000 dilution, mouse anti-GAPDH, Abcam, cat no. 8245) in TBS-T/Odyssey blocking buffer. After three washes of 10 min in TBS-T (0.1% Tween-20), membranes were incubated with secondary antibody (1/5000 dilution; IRDye 680 anti-rabbit, cat no. 926- 68071 and IRDye 800 anti-mouse, cat no. 926-32210; LI-COR) for 30 min in TBS-T/Odyssey blocking buffer. Following three washes in TBS-T and one additional wash in TBS, fluorescent detection was done using an Odyssey infrared imaging system (Odyssey Fc, LI-COR).