A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

• Published in: Analytical biochemistry 2002-09, Vol.308 (2), p.232-238
• Main Authors: Giessauf, Andreas, Flaim, Manuel, Dierich, Manfred P, Würzner, Reinhard
• Format: Article
• Language: English
• Subjects: Animals; Centrifugation, Density Gradient - methods; CHO Cells; Coomassie brilliant blue G; Cricetinae; Crystallization; Crystals; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Indicators and Reagents - chemistry; Rosaniline Dyes - chemistry; Rubella virus - isolation & purification; Rubella virus-like particles; Sucrose gradient ultracentrifugation; Virion - isolation & purification
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(02)00217-8

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3 h, 4 °C) a sharp blue band with crystals (diameter 30–40 μm) was observed (at a density of 1.250 g/ml at 25 °C) in a 30–60% sucrose gradient. Using a combination of SDS–PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS–PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.