Superdex 75 increase 10 300 gl column

The MalE variant T36C/352C is stochastically-labeled with Alexa Flour™ 555 and Alexa Fluor™ 647 dye derivatives as described in Peter et al. and deBoer et al.^{17 (link),84} . The His₆-MalE double variant (200 μg) is incubated with 1 mM DTT and loaded immediately after on 200 μL (wet volume) Ni-Sepharose 6 Fast Flow resin, pre-equilibrated with labeling buffer 1 (50 mM Tris-HCl pH 7.4, 50 mM KCl). After a washing step with 50 column volumes labeling buffer 1, the loaded resin is incubated overnight at 4 ^∘C with 5-fold excess (25 nmol of each fluorophore dissolved in 1 mL of labeling buffer 1. Next, the resin is further washed with 50 column volumes labeling buffer 1 to remove the excess unbound fluorophores. Labeled protein is eluted with 800 μL elution buffer (50 mM Tris-HCl pH 8.0, 50 mM KCl, 500 mM imidazole) and further purified by size-exclusion chromatography (ÄKTA pure system, Superdex 75 Increase 10/300 GL column, GE Healthcare). Protein concentration is determined using the protein extinction coefficient and corrected for direct absorption of the fluorophores at 280 nm. Labeling efficiencies are estimated to be at least 60% for each fluorophore individually and donor-acceptor pairing at least 20%.
Labeled MalE is stored in 50 mM Tris-HCl pH7.4, 50 mM KCl and 1 mgmL⁻¹ bovine serum albumin (BSA) at 4 ^∘C for no more than 3 days. Concentrations ranged between 10 to 100 nM.

Aggregation stability at high concentration was determined by quiescent incubation of samples for 4 weeks at a concentration of 200 mg/mL in 20 mM His/His-HCl, 160 mM Sucrose, 0.02% Poloxamer 188, pH 5.5 at 40 °C, 4 °C, and <−60 °C, respectively. Aggregate levels were determined by size-exclusion chromatography using a Superdex 75 Increase 10/300 GL column (GE Healthcare) with PBS pH 7.4 as the mobile phase.

Recombinant Aβ(M1-42) (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVVIA) and Aβ(M1-40) (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV), here, referred to as Aβ42 and Aβ40, respectively, were prepared by expression in Escherichia coli BL21 (DE3) Gold Strain (Agilent Technologies, Santa Clara, USA) (37 (link)). The resulting inclusion bodies were dissolved in 8 M urea, ion exchanged in batch mode on diethylaminoethyl cellulose resin, lyophilized, and then further purified with a Superdex 75 HR 26/60 column (GE Healthcare, Chicago, USA). Fractions containing the recombinant protein, as determined by SDS-PAGE, were combined and lyophilized again. To ensure we were working with highly purified monomeric species containing extremely low quantities of aggregated forms of the peptides, size exclusion chromatography was carried out directly before the experiments were performed. Aβ40 and Aβ42 solutions were prepared by dissolving the lyophilized peptide in 6 M GuHCl and incubating on ice for 3 hours. The solutions were then purified using a Superdex 75 Increase 10/300 GL column (GE Healthcare, Chicago, USA) at a flow rate of 0.5 ml/min and eluted in 20 mM sodium phosphate buffer (pH 8) supplemented with 200 μM EDTA. The center of the peak was collected, and the concentrations of the peptides were determined from the integration of the absorbance peak using ε₂₈₀ = 1495 L mol⁻¹ cm⁻¹.