Kta avant system

The isolation trials were performed in an ÄKTA Avant system with UNICORN 6 software (GE Healthcare, Wauwatosa, WI, USA) at room temperature. All buffers were filtered through a 0.22 μm pore size membrane and ultrasonically degassed. Butyl-Sepharose 4FF and Octyl-Sepharose 6 FF (GE Healthcare, Wauwatosa, WI, USA) were used as HIC stationary phases. The hydrophobic matrices were packed according to the company guidelines (10 mL gel volume packed into an XK-16 glass column, GE Healthcare, Wauwatosa, WI, USA). The columns were equilibrated with the different tested concentrations of ammonium sulfate in Tris-HCl 10 mM, pH 7.8. LNCaP total protein extracts (500 µL with a protein concentration of ~12 mg/mL) were loaded onto the columns. Isocratic elution at 1.0 mL/min was performed by decreasing stepwise the ammonium sulfate concentration up to 0 M. The pH, pressure, conductivity, and 280 nm absorbance were continuously monitored throughout the entire chromatographic run. The fractions of interest were collected, desalted, concentrated, and stored at 4 °C to further purity and immunoreactivity analysis. The protein content of HIC fractions was measured by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).

An 11 ml lactosyl Sepharose column was connected to an ÄKTA avant system (GE Healthcare). The flow rate was set to 2 ml min⁻¹. The column was equilibrated with 10 column volumes (CV) of MEPBS. The sample was injected and the column was washed with MEPBS (20 CV maximum). The bound protein was eluted with 5 CV MEPBS with 150 mM lactose. During elution, 5 ml fractions were collected. The chromatography run was performed at room temperature, while the fractions were collected at 6°C. Fractions were pooled and concentrated using an Amicon Ultra-15 3 kDa molecular-weight cutoff ultrafiltration spin column (Millipore). The buffer was exchanged for D₂O MEPBS by diluting the concentrated sample (∼5 ml) to 15 ml with fully deuterated buffer and concentrating again, seven times in total, such that the final amount of D₂O in the buffer was 99.9%.
The typical yield of deuterated galectin-3C was 20 mg per litre of cell culture. The protein was filtered through a 0.22 µm filter and stored at 8°C. Its purity was estimated to be >95% by SDS–PAGE (Fig. 1 ▸a) and the degree of deuteration was determined to be ≥92% by mass spectrometry, calculated as a percentage of the possible H→D substitutions.

CEX-HPLC analysis was carried out as described previously [4] (link). Shortly, an ÄKTA Avant system from GE healthcare with a Dionex ProPac WCX-10 (4.0_250 mm) column was used for analysis of recombinant IgG preparations. Mobile phase A was 20 mM sodium acetate, pH 5.0, and mobile phase B was the same with 0.5 M NaCl added. Protein was eluted during a linear gradient from 20% to 80% B at a flow rate of 0.5 ml/min.