Superdex 200 increase

For analysis of mTRAIL and HS oligosaccharide complexes, purified mTRAIL (100 µg) was incubated with HS oligosaccharides (molar ratio 1:1) in 25 mM HEPES, 150 mM NaCl, pH 7.1, at room temperature for 1 hr. For analysis of mTRAIL and low molecular weight/full-length heparin complex, purified mTRAIL (100 ug) was incubated with heparin (molar ratio 1:1) in 25 mM HEPES, 150 mM NaCl, pH 7.1, at room temperature for 1 hr. All complexes were resolved on a Superdex 200 Increase filtration column (Cytiva Lifesciences) using 25 mM HEPES, 150 mM NaCl, pH 7.1, at 4 °C. Presence of a para-nitrophenyl group in the reducing end of the oligosaccharides allows excess oligosaccharides to be visible in the A280 elution profile.

Recombinant proteins (UBE2N and Uev1a, USP5 (163-291a.a), ubiquitin with an N-terminal HA tag, and C-terminally truncated HIV-1 Tat with 2xStrep tag cloned in the pGEX6p-1 vector) were expressed as fusion proteins with an N-terminal GST tag in Escherichia coli BL21 (DE3). The GST-fusion proteins were digested on columns using PreScission protease. The released ubiquitin proteins were collected and further purified by Superdex 200 Increase (Cytiva). TRAF6 full-length protein with a 2xStrep tag was expressed in insect SF9 (Spodoptera frugiperda) cells and purified by Strep-tag affinity chromatography and size exclusion chromatography. C-terminally truncated TRAF6 with N-terminal MBP tag and C-terminal 6xHis tag was expressed in E. coli BL21 (DE3) and purified by using nickel affinity chromatography followed by MBP affinity chromatography.

Samples were collected from the diffusion cells at varying time intervals and the size of the lignin molecules was analyzed by a size exclusion chromatography (SEC) system (HPLC Pump 1515, Autosampler 717plus, Waters, Milford, MA, USA). Two analytical columns packed with Superdex 200 Increase (300 × 10 mm, 9 μm) and Superdex 30 Increase (300 × 10 mm, 9 μm) (Cytiva, Uppsala, Sweden) were used for separation. The columns were operated at ambient temperature and eluted with 0.1 M NaOH solution (analytical grade) as the mobile phase at a flowrate of 0.5 mL/min. Polyethylene glycol (PEG) standards ranging from 200 to 35,000 Dalton (Merck Schuchardt OHG, Hohenbrunn, Germany) were used for calibration. The system was equipped with a Waters 2414 refractive index (RI) detector and a Waters 2487 dual-wavelength UV detector, with absorbances at 280 and 350 nm being used for detection. The RI detector was used for calibration using the PEG standards, whereas—for detection of lignin molecules—the UV detector responses at 280 nm and 350 nm were recorded to monitor the structural changes in the lignin molecules. The delay between UV and RI signals was only a few seconds, which is minimal compared to the analysis time which is about 120 min.