A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals.
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, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees 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.
Giessauf
Andreas
A
Institut für Hygiene und Sozialmedizin, Leopold-Franzens-Universität Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. andreas.giessauf@uni-graz.at
Flaim
Manuel
M
Dierich
Manfred P
MP
Würzner
Reinhard
R
D000818:Animals; D016466:CHO Cells; D002499:Centrifugation, Density Gradient / Q000379:methods*; D006224:Cricetinae; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000737:chemistry*; D012394:Rosaniline Dyes / Q000737:chemistry*; D012412:Rubella virus / Q000302:isolation & purification*; D014771:Virion / Q000302:isolation & purification*
D007202:Indicators and Reagents; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue