Superdex 200 5 150 gl column

Thermostabilities of AdiC variants were determined as described previously [40 (link), 41 (link)]. To test the effect of single mutations on the thermostability of AdiC, 35 μg of purified protein samples in aliquots (70 μl total volume) was incubated at different temperatures for 10 min using a Labcycler gradient PCR machine (SensoQuest GmbH). In order to remove eventual aggregates, a short centrifugation was applied (18,000×g, 30 s, room temperature). The supernatant was then loaded on a Superdex 200 5/150 GL column (GE Healthcare) installed on an ÄKTA Purifier system (GE Healthcare). For each experiment, the column was equilibrated with 1.5 column volumes of SEC-buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.04% (w/v) DDM). The normalization was done by using the untreated sample as reference, which was kept on ice and then analyzed by SEC. For T_m determination, the peak absorbance values at 280 nm were used. Finally, T_m values were calculated by applying the Boltzmann sigmoidal equation in Prism5 (GraphPad Software).

Purified samples of SAMHD1 (2 mg per ml, 50 μl) mixed with a final concentration of 500 µM GTP and 2–4 mM nucleotide analog were applied to a Superdex 200 5/150 GL column (GE Healthcare) pre-equilibrated with SAMHD1 buffer. The UV absorbance at 280 nm was measured as the protein sample eluted from the column, and values were normalized to their respective peak heights.

Thermostability analysis of AdiC variants was performed as described previously [24 (link)]. To test the effect of ligands on the thermostability of AdiC variants, purified samples in aliquots of 35 µg (70 µL total volume and ~11 µM AdiC concentration) were first incubated with the ligands (2 mM; corresponding to 200-fold molar excess) on ice for 10 min. Next, the samples were incubated at different temperatures for 10 min using a Labcycler gradient PCR machine (SensoQuest GmbH, Göttingen, Germany). After a short centrifugation to remove eventual aggregates (18,000× g for 30 s at room temperature), the supernatant was loaded on a Superdex 200 5/150 GL column (GE Healthcare) installed on an ÄKTA Purifier system (GE Healthcare). Prior to injection, the column was equilibrated with 1.5 column volumes of SEC-buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.04% (w/v) DDM, and 2 mM of tested ligand). The SEC peak absorbance values at 280 nm of intact AdiC remaining after denaturation and of untreated sample (reference for normalization; sample kept on ice) were measured and used to calculate the remaining fraction. Remaining fractions against incubation temperatures were plotted (Figure 2) and melting temperatures (T_ms) were calculated by applying the Boltzmann sigmoidal equation in Prism5 (GraphPad Software, La Jolla, CA, USA).

Analytical-scale HPLC was performed with an Agilent 1200 series system on octadecyl carbon chain (C18)–bonded silica columns (5-μm pore size, 4.6 × 250 mm, 218TP54, Vydac). The solvent system was HPLC-grade H₂O (W/0106/PB17, Fisher Scientific) and HPLC-grade acetonitrile (ACN; A/0627/PB17, Fisher Scientific). Buffer A consisted of HPLC-grade H₂O containing 0.1% (v/v) HPLC-grade TFA (T/3258/04, Fisher Scientific), and buffer B consisted of 80% (v/v) ACN in HPLC-grade H₂O containing 0.1% (v/v) TFA.
Mass spectrometry analysis was performed on the Agilent Series 1100 LC-MS system in positive mode of ESI. The solvent system was as follows. Buffer A consisted of LC-MS grade H₂O (W/0112/17, Fisher Scientific) containing 0.1% (v/v) formic acid (06440, Fluka), and buffer B consisted of 80% (v/v) ACN in MS-LC–grade H₂O containing 0.1% (v/v) formic acid. Data analysis was performed with LC/MSD ChemStation software.
SEC-MALS was performed on a Superdex 200 5/150 GL column (GE Healthcare) in SEC-MALS buffer (50 mmol/liter Tris-HCl (pH 7.4), 5 mm MgCl₂, 1 mmol/liter CaCl₂, 100 mm NaCl, and 100 mmol/liter tris(2-carboxyethyl)phosphine). Recombinant troponins were extensively dialyzed against SEC-MALS buffer before experiments. Light scattering and refractive index were measured on a Mini DAWN and OPTILAB DSP (Wyatt Technology, UK), respectively. Data were analyzed with custom-written software.

For serum stability assays, endogenous IgGs were depleted from human male AB plasma (Sigma) by passage through protein G-Sepharose (GE Healthcare). Seldegs were incubated in serum at a concentration of 100 nM at 37 °C for 3 or 5 days. Following incubation, Seldegs were immunoprecipitated using agarose beads coupled to goat anti-human Fc-specific antibody (Sigma). Immunoprecipitated Seldegs were run on 12% SDS–polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore) and membranes incubated with horseradish peroxidase-conjugated goat anti-human Fc-specific (H+L) antibody (Jackson ImmunoResearch). Bound secondary conjugate was detected using Westernsure substrate, followed by scanning with a C-DiGit blot scanner (LI-COR).
Seldegs were also incubated in PBS (Lonza) at 4 °C (30 days) or 37 °C for 5 days, followed by analyses using a Superdex 200 5/150 GL column (GE Healthcare), 12% SDS–polyacrylamide gel electrophoresis and surface plasmon resonance (BIAcore).

Bacterial membranes of 200 mL volume culture were resuspended in 5 mL solubilization buffer (20 mM Tris-Hcl, pH 8, 150 mM NaCl and 1% DDM). After one hour solubilization at 4 °C, insoluble material was discarded by ultracentrifugation for 30 min at 100,000 × g. To avoid different protein/detergent ratios between different strains, 18 mg (2 mg/mL) of membranes were solubilized with 1% DDM in 20 mM Tris-Hcl, pH 8.0, 150 mM NaCl buffer for 1 h at 4 °C. Solubilized MP (100 μL) were loaded on a Superose-6 10/300GL column (GE-Healthcare) for YidC-GFP or onto a Superdex-200 5/150GL column (GE-Healthcare) for YijD-GFP. Size-exclusion chromatography was performed in presence of TBS supplemented with 0.03% DDM. Fluorescence detector (FP-2020, Jasco) was used to measure GFP relative fluorescent intensity with an excitation wavelength of 488 nm and emission wavelength of 512 nm.