Superdex 75 10 300 gl column

Ganoderma lucidum was collected on the campus of The Chinese University of Hong Kong and authenticated by Prof. Shiuying Hu, Honorary Professor of Chinese Medicine, The Chinese University of Hong Kong. DEAE-Sepharose, Mono Q 5/50 GL, Superdex 75 10/300 GL columns and Akta Purifier were bought from GE Healthcare, UK. Primary antibodies against caspase-9, CHOP, IRE1 α, and PARP and secondary antibody for anti-rabbit lgG as well as anti-mouse lgG were purchased from Cell Signaling (Danvers, MA, USA). Primary antibodies against Apaf-1, ATF6, P62, and GAPDH were purchased from Abcam (Cambridge, UK). Primary antibodies against cyclin D1, LC3 and P53 were, respectively, purchased from Millipore (Billerica, MA, USA), Novus (Littleton, CO, USA) and Calbiochem (La Jolla, CA, USA). Click-iT EdU Imaging Kits were purchased from Invitrogen. Image-iT^TM LIVE Green Poly Caspases Detection Kit was obtained from Life Technologies (Carlsbad, CA, USA). WesternBrightTM ECL was purchased from Advansta (Menlo Park, CA, USA).

CyaY was incubated with ammonium iron (II) sulphate ((NH₄)₂Fe(SO₄)₂.6H₂O) at the molar ratio of 7:1 iron to CyaY at 30°C for 1 h before loading on Superdex 75 10/300 GL columns (GE Healthcare). The final concentration for CyaY was 8 mg/ml (0.65 mM) with 4.55 mM Fe²⁺ (a 7:1 ratio of iron to protein). Since the 7:1 iron-to-protein ratio resulted in protein aggregation at protein concentrations higher than 3 mg/ml, 100 μl aliquots of 2 mg/ml CyaY were initially incubated with iron and subsequently concentrated to the required concentration. The sample was always centrifuged for 10 min at 14000 rpm before loading on the columns. The columns were pre-equilibrated with HN100 buffer and operated at a flow rate of 0.5 ml/min at room temperature. Sample elution followed at 280 nm. The size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) experiment was optimized to obtain a well-separated peak for the iron-induced oligomeric species. The best conditions for the experiment included an iron incubation time of 45 min, followed by filtering the sample using a 100 kDa cut-off membrane (Amicon ^® Ultra) to remove aggregates and larger oligomeric particles (> 100 kDa) that could not be separated by the column. The flow-through was then concentrated in a 10 kDa cut-off membrane (Amicon ^® Ultra) to 100 μl before loading onto the SEC column.

Protein samples were diluted to a concentration of 6 mg/mL for the HPV E7 C-terminal domain or 3 mg/mL for the PTP domain and for the complex in a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 2 mM dithiothreitol. SEC-MALS was carried out using a Superdex 75 10/300 GL column (GE Healthcare). The differential refractive index spectra were obtained using Optilab T-rEX (Wyatt Technology), which was combined with high-performance liquid chromatography (Shimadzu) and DAWN HELEOS-II (Wyatt Technology). The weight-average molar mass was calculated using ASTRA 6 software (Wyatt Technology).

Prior to all ThT amyloid aggregation kinetics experiments, the Aβ peptides, after sonication in an ice water bath for 1-3 min in 10 mM NaOH, were subject to one further purification step using size exclusion chromatography (SEC) using a Superdex 75 10/300 GL column (GE Healthcare, USA), to remove aggregated peptides from the monomeric peptides according to previously published protocols (Wallin et al., 2020 (link)). Prepared Aβ₄₀ (12 μM) and Aβ₄₂ peptide (5 μM) solutions were supplemented with ThT (40 μM for Aβ₄₀ and 10 μM for Aβ₄₂) in 20 mM phosphate buffer (pH 7.4 for Aβ₄₀ and pH 8 for Aβ₄₂) and used for the aggregation kinetics measurements. ThT was excited at 440 nm and emission was measured every 2 min at 480 nm in a 96-well plate reader at a 100 μl sample volume (FLUOstar Omega, BMG LABTECH, Germany) under quiescent conditions. Three to five replicates per condition were measured. The data was analyzed with Igor Pro. The phenomenological aggregation parameters were determined from sigmoidal curve-fitting according to Equation 2 (Hellstrand et al., 2010 (link)). $F\left(t\right)=F_0+\frac{A}{1+e^{rmax({\tau }_{\mathrm{½}}-t)}}$ where F₀ is the fluorescence intensity baseline, A is the amplitude, r_max represents the maximum growth rate and τ_½ is the aggregation half time, referred to as the time when 50% of the monomer concentration is depleted.

Truncated human KRAS4B (residues 2–169) was expressed from a modified pET21 bacterial expression vector containing an N-terminal 6×-His purification tag followed by a Tobacco Etch Virus protease cleavage site. SOS^cat (residues 564–1049, pPROEX HTb), and BRAF-RBD (residues 149–232, pET28a) contained a similar vector architecture. All 6xHis-tagged proteins were expressed in Rosetta2 (DE3) cells and purified following the Qiagen Nickel NTA purification protocol and the 6xHis tags were removed using Tobacco Etch Virus protease. For pGEX vectors, proteins were purified following the Glutathione Sepharose 4B purification protocol (Amersham Pharmacia Biotech). The GST-tag was cleaved overnight using thrombin protease while dialyzing in wash buffer. If necessary, the proteins were further purified by size-exclusion chromatography (Superdex-75 10/300 Gl column; GE Life Sciences) and judged greater than 95% pure by SDS-PAGE analysis.

The experiments were performed at 25 °C using a Superdex 75 10/300 GL column (GE HealthCare) connected to a miniDAWN-TREOS light scattering detector and an Optilab T-rEX differential refractive index detector (Wyatt Technology, Santa Barbara, CA, USA). The column was equilibrated in 50 mM BisTris pH 6.7, 50 mM NaCl, 1 mM TCEP and 0.5 mM EDTA buffer filtered at 0.1 µM, and the SEC-MALS system was calibrated with a sample of Bovine Serum Albumin (BSA) at 1 mg/mL. Samples at 1.5 mM, 0.6 mM, and 0.7 mM were prepared for V2R-Cter, GHSR-Cter, and β2AR-Cter, respectively. For each GPCR-Cter, 40 µL of sample were injected at 0.5 mL/min. Data acquisition and analyses were performed using the ASTRA software (Wyatt).