Superose 6 size exclusion column

The TIM/TIPIN protein complex was further purified using size exclusion chromatography to verify the stability of the complex. The samples were purified on a Superose 6 size exclusion column (GE Healthcare, Marlborough, MA, USA) in 50 mM HEPES, pH 7.8, 150 mM NaCl, and 0.5 mM TCEP. The fractions corresponding to the TIM/TIPIN complex were eluted, collected into a new Eppendorf tube, and loaded onto an SDS–polyacrylamide gel for further SDS–PAGE and stained with Coomassie blue stain solution and the presence of both proteins was confirmed by immunoblot (IB) analysis using TIM and TIPIN antibodies.

Mitochondria (1 mg protein) were suspended in 500 µl digitonin containing buffer (20 mM Tris–HCl, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 10% [vol/vol] glycerol, 1 mM PMSF, 1% digitonin). After solubilisation for 30 min on ice a clarifying spin was performed (10 min at 12,000 × g, 4°C). Cleared lysates were subjected on Superose 6 size exclusion column (GE Healthcare, Piscataway, NJ, USA; elution buffer 20 mM Tris, pH 7.4, 150 mM NaCl, 5% [vol/vol] glycerol and 0.075% [wt/vol] digitonin). Fractions were analyzed by SDS-PAGE and immunoblotting. To calibrate the column, a mixture of thyroglobulin, catalase and carboanhydrase was subjected to the same procedure.

Nuclear extract of six brains from 4-week-old male C57BL/6 mice was dialyzed against buffer CB (50 mM Tris-HCl pH 7.9, 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 10% glycerol, 0.1 mM PMSF, 1 mM DTT) and loaded onto a 1 ml Source15Q anion exchange column (GE Healthcare) on an Äkta Explorer FPLC system (GE Healthcare). After washing with 10 column volumes (CV) buffer CB, proteins were eluted with a 15 CV linear gradient from 100 to 500 mM NaCl in buffer CB. 0.3 ml fractions were collected and subjected to immunoblotting using antibodies against different ChRFs and HDACs. Source15Q fractions containing peak amounts of the analyzed proteins (200–280 mM NaCl) were pooled, applied to a 100 ml Superose 6 size exclusion column (GE Healthcare) and eluted with buffer CB. Two milliliters fractions were collected and proteins were precipitated by addition of 20% (final) trichloroacetic acid (TCA) and incubation for 20 min on ice. Precipitates were collected by centrifugation at 17000 × g for 15 min, washed twice with acetone, dried on ice and dissolved in 1 × SDS loading buffer (75 mM Tris-HCl, pH 6.8, 0.6% SDS, 15% glycerol, and 1.075 M β-mercaptoethanol) for subsequent SDS-gel electrophoresis and western blotting.

NCI-H526/COR-L311 nuclear extracts were obtained using NE-PER nuclear extraction kit (Thermo Scientific) and dialyzed against FPLC buffer (20 mM Tris-HCl, 0.2 mM EDTA, 5mM MgCl2, 0.1 M KCl, 10% (v/v) glycerol, 0.5 mM DTT, 1 mM benzamidine, 0.2 m MPMSF, pH7.9). 5mg of nuclear protein was concentrated in 500 μl using a Microcon centrifugal filter (Millipore) and then applied to a Superose 6 size exclusion column (10/300 GL GE Healthcare) pre-calibrated using the Gel Filtration HMW Calibration Kit (GE Healthcare). 500 μl elute was collected for each fraction at a flow rate of 0.5ml/min, and eluted fractions were subjected to SDS-PAGE and western blotting.

HeLa cell nuclear extracts or FLAG-CEP131-containing protein complexes were applied to a Superose 6 size exclusion column (GE Healthcare) that had been equilibrated with dithiothreitol-containing buffer and calibrated with protein standards (Amersham Biosciences). The column was eluted at a flow rate of 0.5 ml min⁻¹ and fractions were collected.

All serum samples were analyzed for ALAT, creatinine, and fructosamine concentrations on an automated chemistry analyzer (Abbott Architect c4000, Abbott Park, IL, USA) at the Clinical Pathology Laboratory, University Animal Hospital, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Lipoprotein profiles were obtained at the department of Medical Biosciences, Umeå University by utilizing an automated HPLC system (Elite LaChrom, Hitachi, Krefeld, Germany) with a Superose 6 size-exclusion column (GE Healthcare, Uppsala, Sweden). Plasma samples were diluted 1:16 in elution buffer that consisted of 10 mM Tris, 150 mM NaCl and 0,02% NaN₃, and injected into the column. On-line measurements of triglyceride and cholesterol concentrations were performed using appropriate reagents (Roche, Basel, Switzerland). The reagents were diluted 1:2 with lab grade water prior to analyses. As a standard for lipoprotein profiles, a human plasma sample with a known lipid concentration was used. All data was processed using the EZChrom Elite software (Agilent Technologies, Boeblingen, Germany).
Free fatty acids were measured with the MaxDiscovery^™ Non-esterified fatty acids (NEFA) Assay Kit (Bioo Scientific, Austin TX, US), at the Clinical Sciences laboratory, Swedish University of Agricultural Sciences, Uppsala, Sweden.