2110 fraction collector

A total of 10 to 40% sucrose gradients were prepared (solvated in 25 mM HEPES Tris, 100 mM potassium glutamate, 20 mM magnesium acetate, 14 mM β-mercaptoethanol, and 0.5% Tween 20) in polyclear centrifuge tubes (Seton Scientific) using a Gradient Master instrument (BioComp Instruments) the day of culture harvest and kept at 4°C until lysates were prepared. A total 200 μL of normalized lysates were added to the tops of the gradients. Cellular components were separated in the gradients using a Beckman Coulter Optima L-90K ultracentrifuge with a SW41 Ti rotor at 35,000 RPM for 3.5 h at 2°C. The gradients were then fractionated using a BioComp Piston Gradient Fractionator with a Bio-Rad 2110 Fraction Collector while absorbance was measured at 258 nm.

293T cell lysates (0.8 ml) were fractionated using a Sephacryl S-100 column (GE Healthcare). Fraction sizes of 500 μl were collected using a Bio-Rad 2110 fraction collector. Samples were analyzed by immunoblotting after SDS-PAGE separation.

Crude fucoidan was purified by fractionation using anion-exchange chromatography. Crude fucoidan (2 g) was dissolved in 20 mL of Tris-HCl buffer (0.05 M, pH 7.4) and applied to a column (25 cm × 4 cm) of DEAE–Sephadex A-25 (Pharmacia Ltd.) equilibrated with Tris-HCl buffer connected to a Bio-Rad 2110 fraction collector. The first fraction was eluted with deionized water at a flow rate of 40 drops per tube, followed by NaCl elution at increasing concentrations (1 and 2 M) until the absence of a positive reaction for the presence of sugars in the test tubes when using the phenol–sulfuric acid method according to Dubois et al. (27 (link)). Briefly, test tubes containing the eluted samples were transferred (1 mL) into more robust glass test tubes (5 mL). Then 0.05 mL 80% phenol and 2.5 mL concentrated H₂SO₄ were added to each test tube and mixed thoroughly. Test tubes were placed on a rack and heated in a 35°C water bath for 20 min. The absorbance was measured at 480 nm (Ultrospec 2100) for any indication of sugars and uronic acids (27 (link)). Each carbohydrate-positive fraction was pooled together, dialyzed for 72 h (MWCO 12–14,000) in deionized water with a water change daily and then lyophilized.