Hiprep deae ff 16 10 column

At the end of the cultivation, 600 mL of the culture was centrifuged to collect the algal sludges. The sludges were placed in 20 mL of 20 mM Tris-HCl buffer (pH 8.0). Repeated freezing and thawing 3−4 times to extract took place until no red in algae cells. The extract was filtered using 0.45 μm microporous filter membrane (Tianjin jinteng Experimental Equipment Co., Ltd., Tianjin, China) and then loaded onto a HiPrep DEAE FF 16/10 column (GE Healthcare, Chicago, IL, USA) to elute with a gradient of 0–1.0 M NaCl. The purified eluent was concentrated using ultrafiltration centrifuge tubes (10,000 MWCO) (Millipore Corporation, Bedford, MA, USA) and then loaded onto a Superdex 200 Increase 10/300 GL column (GE Healthcare, Chicago, IL, USA) to further purify with elution buffer (200 mM NaCl and 20 mM Tris-HCl (pH 8.0)). The absorbance of phycoerythrin at 280 nm and 545 nm were measured using TU-1810 UV-Visible spectrophotometer. The optical density at the wavelength of 280 nm and 545 nm were used to represent the concentration of crude total protein and B-phycoerythrin, respectively [30 (link),31 (link),32 (link),33 (link)]. The purity of B-phycoerythrin was expressed by the following equation:
where B-PE is B-phycoerythrin. OD₂₈₀ and OD₅₄₅ are the optical density values at 280 nm and 545 nm, respectively.

A. nidulans transformants expressing AnLPMO9C, AnLPMO9F and AnLPMO9G were grown in Erlenmeyer flasks (2 L) containing 200 mL MM pH 6.5 supplemented with 1% (wt/vol) glucose and 2% (wt/vol) maltose. Cultivation was performed for 36 h under static conditions at 37°C. Cultivation supernatants were centrifuged (5.000g, 40 min, 4°C), filtered (0.45 um) and loaded onto a HiPrep DEAE FF 16/10 column (GE Healthcare), equilibrated in 50 mM Tris–HCl buffer pH 7.0. Proteins were eluted using a linear gradient up to 1 M NaCl in the same buffer. Fractions containing a protein band with the expected theoretical MW were pooled and concentrated. The concentrated sample was applied to a HiTrap Phenyl HP 1 mL column (GE Healthcare) and eluted in the same buffer with a linear gradient from 1.5 – 0.0 M ammonium sulfate. Fractions containing a protein band with the expected theoretical MW were pooled. Partially purified enzymes were incubated with Cu(II)SO₄ (3:1 molar ratio of copper:enzyme) overnight at 4°C. Then, samples were concentrated, washed in 50 mM MES buffer pH 6.0 and applied to a Superdex 75 column (GE Healthcare) equilibrated in the same buffer. Eluted fractions were analyzed in SDS-PAGE and those presenting the purified enzyme were pooled. Protein concentration was determined by reading A₂₈₀ and considering the theoretical MW and molar extinction coefficient of each enzyme.

Acrylamide, methylene-bis-Acrylamide, N,N,N′,N′-tetramethylethylenediamine, and ammonium persulfate for sodium dodecyl sulfate polyAcrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad. Blue dextran, thyroglobulin, bovine catalase, bovine serum albumin, and lysozyme were purchased from Sigma. HiPrep DEAE FF 16/10 column, Superdex 200 10/300 GL column, and Resource Q column were purchased from General Electrics. All other chemicals were of the best purity available.

All enzymes were purified by two chromatography steps using the HiPrep™ DEAE FF 16/10 column (GE Healthcare), followed by a HiLoad™ 16/600 Superdex™ 200 pg column (GE Healthcare), as previously described [14 (link)]. Purification was evaluated by SDS-PAGE and protein concentration was determined by reading absorbance at 280 nm, using the molar extinction coefficient [31 (link)] calculated from the amino acid composition (http://web.expasy.org/protparam/) [68 ].

Recombinant human wild type alpha-synuclein was overexpressed by E. coli BL21(DE3) with plasmid pET28a. Cells were grown in LB medium in the presence of 50 µg/mL Kanamycin and protein expression was induced by 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cell pellet was resuspended in Tris buffer (25 mM Tris-HCl, pH 7.4) and lysed by sonication. After centrifugation at 30,000 × g for 45 min at 4 °C, the supernatant was boiled to remove most E. coli proteins. After centrifugation at 30,000 × g for another 60 min at 4 °C, the supernatant was loaded onto HiPrep DEAE FF 16/10 column (GE Healthcare). A gradual sodium chloride gradient was chosen and applied to elute the target protein. After SDS-PAGE gel analysis, fractions containing the target protein were desalted by HiPrep™ 26/10 Desalting column. The desalted solution was loaded onto HiPrep 16/60 Sephacryl S-100 column (GE Healthcare) for further purification, with 25 mM Tris-HCl, pH 7.4 buffer as the running buffer. Fractions were analyzed by SDS-PAGE gel, and targeted protein were collected, concentrated and stored at −20 °C. The concentration of alpha-synuclein was determined by UV-1800 spectrophotometer (SHIMADZU) with the extinction coefficient of 5960 cm⁻¹ M⁻¹ at 276 nm.

Overnight 5 mL liquid cultures of yeast strains in synthetic defined dropout media were diluted into 50 mL of fresh media and grown at 30 ^oC for 18–24 h. Cells were resuspended in 6 mL PBS buffer and lysed using glass beads, and then sodium chloride and PEG-8000 were added to the soluble fraction to a final concentration of 0.5 M and 8%, respectively. After sitting for 15 min on ice, the precipitate was isolated, redissolved in 2 mL PBS buffer, and purified by size exclusion using a HiPrep 16/60 Sephacryl S-500 HR column (GE Healthcare) in PBS buffer (1 mL/min) on an AKTA Explorer (Amersham Biosciences). The encapsulin fractions were concentrated using Amicon Ultra-15 Centrifugal Filter Units with Ultracel-100 membrane (Millipore), then diluted in 2 mL of 20 mM Tris buffer at pH 8. Ion-exchange chromatography using a HiPrep DEAE FF 16/10 column (GE Healthcare) resulted in the fully purified encapsulin sample for further analysis. The gradient used for ion-exchange was as follows: 100% A for 0–100 mL, 100% A to 50% A + 50% B for 100–200 mL, 100% B for 200–300 mL, 100% A for 300–400 mL; where A is 20 mM Tris pH 8, B is 20 mM Tris pH 8 with 1 M NaCl (flow rate: 3 mL/min). Examples of purification chromatographs can be found in Supplementary Figs. 13 and 14.