Superose 6 10 300 gl gel filtration column

For purification of native yeast Nup84-subcomplex (KWY6349) yeast membranes were prepared and extracted essentially as described in “Preparation of crude membrane extracts” except that 0.05% CHAPS was used as a membrane extraction detergent and the extract was used for protein purification straight after vortexing with glass beads. The extract was incubated with IgG-coupled Sepharose beads for 2 hours at 4°C. The beads were washed with TEV-Elution Buffer (50 mM HEPES pH 7.5, 300 mM KCl, 10mM MgCl2, 0.1mM EDTA, 5mM β-mercaptoethanol, 0.05% CHAPS) in Poly-prep chromatography columns (Bio-Rad). Bound Nup84-subcomplex was cleaved off from the beads with TEV protease and separated from beads by passing through 0.45 μm Ultrafree-MC HV centrifugal filter (Merck Millipore). To analyze the quality of Nup84-subcomplex, the prep was subjected to gel filtration analysis on Superose 6 10/300 GL gel filtration column (GE Healthcare) equilibrated with TEV-Elution Buffer without CHAPS.

A Superose 6 10/300 GL gel filtration column (24 ml bed, GE Healthcare) was equilibrated with F-buffer [0.1 M KCl, 2 mM 2 mM MgCl₂, 0.2 mM DTT, 20 mM HEPES (pH 7.5)], and proteins at 1.0 mg/ml (500 µl) were applied and eluted with the same buffer. The eluates were collected in fractions of 0.5 ml each, and analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue R-250. Gels were scanned by an Epson Perfection V700 Photo Scanner at 300 dots per inch, and band intensity was quantified using Image J. Apoferritin (443 kDa) (A3660, Sigma-Aldrich), β-amylase (200 kDa) (A8781, Sigma-Aldrich), alcohol dehydrogenase (150 kDa) (A8656, Sigma-Aldrich), and Bovine serum albumin (66 kDa) (A8531, Sigma-Aldrich) were used as molecular mass standards.

Glycosaminoglycan polysaccharides (4 mg) were dissolved in 0.5 ml of ammonium bicarbonate (100 mM) for 24 hr at 4°C and then centrifuged at 12000g for 10 minutes prior to application to a Superose 6 10/300GL gel filtration column (GE Healthcare, Little Chalfont, UK) running at a flow rate of 0.75 ml/min in the same eluent. Samples eluting from the column at room temperature passed through an in-line DAWN HELEOS-II laser photometer with a 658 nm laser (Wyatt Technology Corp., Santa Barbara, USA), WyattQELS detector model number Q-03 and an Optilab rEX refractometer; a refractive index increment (dn/dc) of 0.16 mlg⁻¹ was used throughout.^{28 (link)} Light scattering and concentration during elution were recorded using ASTRA software (v5.3.4.13). Multi-angle laser light scattering was used to determine weight-averaged molecular masses (M) and radius of gyration (R_g) using analysis modules within the ASTRA software. For molecules too small to accurately determine R_g, quasi-elastic light scattering was used to measure hydrodynamic radii (R_h), again using analysis modules within the ASTRA software. Conversion of calculated R_g to R_h was achieved using the formula R_g = 1.5R_h, derived previously for a random coil.^{29 (link)}