Monoq 10 100 gl column

For the synthesis of 5–50 mg polymer, enzymes were incubated overnight at 37 °C in 3–30 ml of assay buffer (50 mM Tris, pH 8.0, 10 mM MgCl₂, 1 mM DTT (dithiothreitol)) supplemented with 5 mM of each substrate. In vitro-synthesized polymer was purified by AEC using a Mono Q 10/100 GL column (GE Healthcare) as described previously^{30 (link)} with the minor adjustment that polymer-containing fractions were either dialyzed against water or thoroughly washed using an Amicon centrifugal device (30,000 molecular weight cutoff; Merck).

G proteins and scFv16 for structural study were prepared as previously described (15 (link), 31 (link)). For GTP turnover assay, we further purified G proteins using mono Q 10/100 GL column (GE healthcare) to ensure the 1:1:1 stoichiometry of Gα:Gβ:Gγ chains.

E. coli strain BL21(DE3)pLysS (Invitrogen, Inc.) was transformed with plasmid pET21a-gp32 (GENEWIZ, Inc.) encoding gp32 under the control of the bacteriophage T7 gene 10 promoter. Single colonies of the resulting transformants were used to inoculate 5 l LB broth containing 50 μg/ml ampicillin, cultures were incubated at 37 °C with shaking until OD₆₀₀ = 0.8, cultures were induced by addition of IPTG to 0.4 mM, and cultures were incubated 3 h at 37 °C. Then, cells were harvested by centrifugation (5422 × g; 10 min at 4 °C), resuspended in 70 ml buffer G (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 5% glycerol, 1 mM EDTA, and 1 mM DTT), and lysed using a JN-02C cell disrupter (JNBIO, Inc.). The lysate was centrifuged (24,792 × g; 45 min at 4 °C), and the supernatant was loaded onto a 5 ml column of HiTrap Heparin HP (GE Healthcare, Inc.) equilibrated in buffer G and eluted with a 100 ml linear gradient of 0.1–1 M NaCl in buffer G. The sample was further purified by anion-exchange chromatography on a Mono Q 10/100 GL column (GE Healthcare, Inc.; 160 ml linear gradient of 0.1–1 M NaCl in buffer G). Fractions containing gp32 were concentrated to 5 ml using an Amicon Ultra-15 centrifugal filter (10 kDa MWCO; Merck Millipore, Inc.) and applied to a HiLoad 16/600 Superdex 200 column (GE Healthcare, Inc.) equilibrated in buffer E, and the column was eluted with 120 ml of the same buffer.

The mRNA sequence of the dimerization and DNA-binding domain of human MYC was cloned into the pETM11 (EMBL-Heidelberg) vector. The protein was expressed as an N-terminally His-tagged protein in ArcticExpress (DE3)RIL cells (Agilent Technologies, Santa Clara, USA) after an OD of 0.8 was reached by induction with 0.1 mM isopropyl-β-thiogalactoside (IPTG) at 14°C for 18 hr. The protein was purified to homogeneity by metal affinity chromatography (Ni-NTA, Thermo Fisher Scientific) followed by anion exchange chromatography (AEC). AEC was performed using a MonoQ 10/100 GL column (GE Healthcare, Chicago, USA) equilibrated with 20 mM HEPES pH 8.0 and 0.25 M NaCl. The protein did not bind to the column but eluted in the flow through. Size exclusion chromatography was performed using a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) to confirm its monomeric state and exclude aggregation. The protein was concentrated after AEC to 0.59 mg/ml based on the calculated extinction coefficient using ProtParam (SwissProt) and then flash frozen adding 10% glycerol for storage.

The periplasmic fraction of the T. paradoxus ARh1 cells grown on thiocyanate was isolated as described in [3 (link)]. CytC552 purification involved three stages. The first step included anion-exchange chromatography on a MonoQ 10/100 GL column (GE Healthcare Life Sciences) pre-equilibrated with 25 mM MOPS, pH 7.5. The proteins were eluted with a 0–1 M linear gradient of NaCl in the same buffer. The CytC552-containing fractions were pooled and loaded on affinity HisTrap HP 5 mL column (GE Healthcare Life Sciences, Chicago, IL, USA) pre-equilibrated with the 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole buffer, pH 8. Imidazole gradient from 20 to 500 mM in the same buffer was applied and fractions containing CytC552 were pooled, concentrated and loaded on the gel filtration column Superdex 75 10/300 GL (Amersham Biosciences, Chicago, IL, USA) pre-equilibrated with the 50 mM Tris-HCl, 150 mM NaCl buffer, pH 8. CytC552-containing fractions were analyzed by SDS-PAGE and MALDI-TOF-MS methods.

Sequences of Schistosoma proteases were obtained via BLAST and used to manually identify homologs found using the Schmidtea mediterranea database (SmedDb; http://planaria.neuro.utah.edu) [4 (link)]. Hits were checked via BLAST; reciprocal best hits were scored as putative homologs. The major cathepsin B homolog was isolated from planaria lysate (~50 planarians in lysis buffer; 100mM Tris pH 7.5, 200mM NaCl, 1% NP-40, 0.1% SDS, 1XTBS). Lysate was separated on a MonoQ 10/100 GL column (GE Healthcare Life Sciences) and assayed for protease activity with Z-RR-AMC (BACHEM). Fractions of cathepsin B activity were pooled and separated on a Superdex 200 10/300 GL column (GE Healthcare Life Sciences) which had been tested with a Gel Filtration Calibration Kit LMW (GE Healthcare) to determine at what volume the cathepsin B homolog would elute. The resulting samples with proteolytic activity were run on a 10% SDS-PAGE gel, silver stained, and cut into bands for analysis on PE-Biosystems Voyager Elite STR MALDI-TOF. The band corresponding to the most proteolytic activity and correct size was determined to be the cathepsin B homolog identified by SmedDb.