Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation.
A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.
Ali
Akhtar
A
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA. aali@noble.org
Roossinck
Marilyn J
MJ
D015153:Blotting, Western; D017795:Bromovirus / Q000302:isolation & purification* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D009029:Mosaic Viruses / Q000302:isolation & purification* / Q000648:ultrastructure; D010935:Plant Diseases / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D013997:Time Factors; D014771:Virion / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure
D012367:RNA, Viral