Gradient mini protean tgx pre cast gel

Plasmids were transformed into BL21-DE3 cells (Agilent) and induced for expression at 0.7–0.8 optical density with 1 mM isopropyl β-d-1-thiogalactopyranoside typically for 3 h. Proteins were purified from the cell lysate by nickel-affinity chromatography (NAC, step 1). The bound fraction was subjected to isocratic fractionation on Superose-12 column (step 2, Cytiva Life Sciences) and HRV-3C protease cleavage (step 3, purchased from Sigma-Aldrich) or TEV protease (produced in-house^{48 (link)},) overnight at 4 °C followed by repeating NAC and step 2 in a final buffer of 25 mM Tris-HCl, pH 7 or 7.6, 150 mM NaCl and 1 mM TCEP (buffer A). The full-length wild-type (MPro^WT) was expressed and purified similar in strategy to that described previously except for substituting the fusion partner GST with maltose binding protein (MBP) followed by a 36 amino acid spacer sequence corresponding to the immunoglobulin binding domain B1 of protein G (ΔGB1). Peak fractions were pooled and concentrated to the desired concentration and stored in aliquots at −30 °C and for long term storage at −80 °C. Purity was verified both by SDS-PAGE on 4–20% gradient mini-protean TGX precast gel (Bio-Rad) and electrospray ionization mass spectrometry.

Cells were washed, lysed with lysis buffer [250 mM NaCl, 10% glycerol, 2mM EDTA, 0.5% IGEPAL (Sigma-Aldrich), 50 mM HEPES (Sigma-Aldrich)], and then centrifuged at 13,000 RPM for five minutes. Afterwards, 2x Laemmli sample buffer (Bio-Rad Laboratories, Hercules, California) was added to the lysate for a 1:1 dilution and the sample was denatured at 95 °C for five minutes. Then, the sample was loaded onto a 4–20% gradient Mini-PROTEAN TGX precast gel (Bio-Rad Laboratories) and electrophoresed at 100 V for 70 minutes, before being transferred to a PVDF membrane (Bio-Rad Laboratories) via wet transfer. Membranes were blocked with 5% dry milk in TBST [10 mM Tris-HCl, 100 nM NaCl, 0.1% Tween 20] for 1 hour and then incubated with an anti-GFP primary antibody (Abcam) at a 1:5000 dilution or a GAPDH primary antibody (Santa Cruz, Dallas, TX) at a 1:3000 dilution overnight at 4 °C. Afterwards, the membrane was rinsed three times with TBST and incubated with the appropriate species-specific enzyme-conjugated secondary antibody (Life Technologies), for 1 hour at room temperature. Membranes were later developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The resulting signal was detected using digital imaging (Bio-Rad Laboratories).

With the purpose of distinguishing between glycoproteins that have not reached the mid-Golgi and folded, processed, mature glycoproteins, cell lysates containing 30 µg of total protein were subjected to overnight incubation at 37 °C with 5 µL of Endo-H or 2.5 µL of Endo-F (Roche, Basel, Switzerland) upon denaturing the samples for 5 min. at 95 °C in Endo-H or Endo-F denaturing buffer (Endo-H Buffer: buffer sodium citrate 50 mM pH 5.2, PMSF 0.5 mM, SDS 0.1%, Triton X 100 0.5%, mercaptoethanol 0.1 M; Endo-F Buffer: Buffer sodium phosphate 100 mM pH 7.2, EDTA 10 mM, Triton X 100 0.1%, SDS 0.1%, mercaptoethanol 1%). Digested and undigested samples were then separated on a 4%–20% gradient mini-protean TGX pre-cast gel (BioRad, Hercules, CA, USA) and western blot analysis was performed using anti-GBA 2E2 antibody, as described above.