Deae sephacel

The 20S proteasome was purified as described previously (Groettrup et al, 1995 (link); Schmidtke et al, 1996 (link); Basler & Groettrup, 2012 ). In brief, cell pellets from LCL721.45 cells were collected and lysed with 100 mM KCl + 0.1% Triton X-100. The suspension was homogenized with a Dounce homogenizer and subsequently centrifuged for 30 min at 30,600g and 4°C. The supernatant was mixed with the anion exchanger DEAE Sephacel (GE Healthcare) and incubated overnight at 4°C while rotating. The next day, the DEAE mix was column purified and eluted with 500 mM KCl. (NH₄)₂SO₄ was added (35% of total volume), and samples were centrifuged twice for 20 min at 17,211g at 4°C. The resulting pellet was resolved in 100 mM KCl and incubated on ice for 60 min. Afterward, the dissolved pellet was added on a sucrose gradient (15–40%) and centrifuged for 16 h at 274,355g at 4°C.
After centrifugation, the sucrose gradient was separated into fractions of 700 μl and tested for proteasomal activity and further purified via a Resource Q column and fast protein liquid chromatography. Successful isolation of the 20S proteasome was confirmed with activity assay and SDS–PAGE.
Recombinant FAT10 was purified from transfected Escherichia coli BL21(DE3) CodonPlus cells (Stratagene) as described before using Ni-affinity chromatography and size-exclusion gel filtration (Aichem et al, 2018 (link)).

A small polyprep column from BioRad was packed with 500 µL of DEAE-Sephacel (GE-Healthcare) and the bed was calibrated with 5 mL of diethylaminoethyl DEAE pre-wash buffer (50 mM NaOAc/150 mM NaCl with 0.1% Triton X-100 at a pH of 6.0). A total of 1 mg of crude GAGs was dissolved in 500 µL of deionized water, with 250 µL of this solution was loaded onto the column. After this, the column was washed with 5 mL of DEAE washing buffer (50 mM NaOAc/150 mM NaCl with at a pH of 6.0). The GAGs were eluted by adding 2.5 mL of DEAE elution buffers (1–50 mM NaOAc/1M NaCl pH 6.0, 2–50 mM NaOAc/2M NaCl pH 6.0 and 3–50 mM NaOAc/3M NaCl pH 6.0). Each fraction was collected separately. All samples had salt removed using the PD-10 column.

Lipase type VII from C. rugosa was obtained from Sigma Chemicals Co. (St Louis, MO, USA), tributyrin and triacetin from Fluka (Deisenhofen, Germany), and sodium deoxycholate from Amresco (Solom, OH, USA). Gels for protein purification, DEAE-Sephacel, Phenyl-Sepharose CL-4B, Sephacryl HR 100, and the Sephacryl S200 column were from GE Healthcare (Piscataway, NJ, USA). All chromatographic steps were performed on a fast protein liquid chromatography (FPLC) system (Pharmacia Biotech, Sweden).

Heparan sulfate was isolated from the cells as described previously (40 (link)). Briefly, 1 × 10⁶ cells were digested with 0.4 μg/ml Pronase (Sigma-Aldrich) at 37 °C for 16 h and crude GAGs were isolated by anion exchange chromatography using DEAE-Sephacel (GE Healthcare). The columns were washed with 0.25 m NaCl and the glycosaminoglycans were eluted with 2 m NaCl. Heparan sulfate chains were depolymerized with heparin lyase I, II, and III (2 milliunits/ml of each) and unsaturated disaccharides were analyzed using glycan reductive isotope labeling liquid chromatography/mass spectrometry techniques as described elsewhere (40 (link)).