Superose 6 increase 10 300 gl

The size-exclusion chromatography (SEC) was conducted to analyze the aggregation of wild-type PRA and PRA mutants. Briefly, Superose^TM 6 increase 10/300GL (GE healthcare) were connected to ÄKTA pure 25 chromatography system (GE healthcare) and balanced using elution buffer (0.1 mol NaCl, 0.05 mol NaH₂PO₄. 2H₂O, pH 7.5) first. Next, 1 mg wild-type PRA and PRA mutants were inputted and eluted using same elution buffer with elution speed of 0.5 mL/min. The absorbances peaks (mAU 280 nm) for wild-type PRA and PRA mutants were monitored and recorded for further analysis.

The WT protein and NxT PNGS mutants were analyzed by negative stain EM as previously described (Cottrell et al., 2020 (link)). For the RM20E1 complexes, SOSIP trimers were incubated with 6-fold molar excess per protomer Fab at RT overnight. The complexes were purified using SEC on a Superose^™ 6 Increase 10/300 GL (GE Healthcare) column. Samples were imaged and analyzed as previously described (Cottrell et al., 2020 (link)).

Soluble fractions obtained during the ThT kinetic measurements (Fig. 8c, d) were directly injected into a Superose 6 Increase 10/300 GL (GE Lifesciences, #29-0915-96). All runs were performed in 10 mM Tris-Cl (pH 7.4) at a flow rate of 0.7 mL min⁻¹, and the absorbance was monitored at 280 nm using a ÄKTA Prime System (GE Lifesciences). The column was previously calibrated using thyroglobulin, 670 kD; γ-globulin, 158 kD; ovalbumin, 44 kD; myoglobin, 17 kD; and vitamin B12, 1.35 kD (Bio-Rad, #151-1901).

The hrCN PAGE protocol was adapted from Lemaire et al. (2018) (link). Glycerol was added to the sample at a final amount of 20% (v/v). Ponceau S at a final concentration of 0.001% (w/v) served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM tricine, 15 mM Bis-Tris/HCl, pH 7.0, 0.05% (w/v) sodium deoxycholate, 2 mM DTT, and 0.01% (w/v) dodecyl maltoside, whilst the anode buffer contained 50 mM Bis-Tris/HCl, PH 7.0, 2 mM DTT. An 8–15% linear polyacrylamide gradient gel was used, and electrophoresis was run under a N₂/CO₂ (90:10%) atmosphere with a constant 40 mA current (PowerPac^™ Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^™ Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^™ Unstained Protein Standard (Thermo Fischer Scientific, Driesch, Germany).
The determination of the oligomeric state by gel filtration was performed in triplicate on a Superose 6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.6, 2 mM DTT, 10% (v/v) glycerol at a flow rate of 0.4 ml/min, and in an anaerobic Coy tent containing an N₂/H₂ (97:3%) atmosphere. High molecular weight range gel filtration calibration kit (GE Healthcare, Munich, Germany) was used as the protein standard.

The hrCN PAGE protocol was adapted from Lemaire et al. (2018) [24 (link)] Glycerol was added to the sample at a final amount of 20% v/v. Ponceau S at a final concentration of 0.001% w/v served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7, 0.05% w/v sodium deoxycholate and 0.01% w/v dodecyl maltoside, while the anode buffer contained 50 mM Bis-Tris/HCl buffer pH 7. A 5 to 15% linear polyacrylamide gradient gel was used and electrophoresis was run with a constant 40 mA current (PowerPac^TM Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^TM Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^TM Unstained Protein Standard (ThermoFischer Scientific, Dreieich, Germany).
Determination of the oligomeric state by gel filtration was performed on a Superose6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.4, 2 mM DTT, 10% glycerol at a 0.4 mL/min flow rate. High Molecular Weight range Gel Filtration Calibration Kit (GE Healthcare, Munich, Germany) was used as the protein standard.