Purification of tomato yellow leaf curl geminivirus.
Attempts were made to find a good purification procedure for tomato yellow leaf curl virus (TYLCV), a dangerous and continuously spreading whitefly-transmitted germinivirus, up to now only partially purified. Electron microscopy, serology and spectrophotometry were used to evaluate different procedures. The scheme finally adopted was the following: collect leaves and stems from Nicotiana benthamiana graft-infected 45-60 days previously (5-10 g/plant); homogenize with 0.5 M phosphate buffer pH 6 containing 2.5 mM NaEDTA, 10 mM Na2SO3, 0.1% 2-mercaptoethanol, 1% Triton X-100 and 0.1% Driselase (3-4 ml of buffer for each g of material); incubate overnight on ice with gentle agitation; filter; emulsify with 15% cold chloroform; centrifuge at low speed; ultracentrifuge supernatant; resuspend pellets in 0.5 M phosphate buffer pH 7 containing 2.5 mM NaEDTA; centrifuge at low speed; repeat resuspension of the pellets and low-speed centrifugation; ultracentrifuge the pooled supernatant on a Cs2SO4 gradient (e.g. for 5 h at 41,000 rpm); collect the virus band and dialyse or ultracentrifuge the virus. The virus yield was 5-10 mg per kg of tissue.
Luisoni
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Istituto di Fitovirologia applicata, Consiglio Nazionale delle Ricerche, Torino, Italy.
Milne
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RG
Vecchiati
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D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D005656:Fungal Proteins; D017791:Geminiviridae / Q000276:immunology / Q000302:isolation & purification*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D018551:Solanum lycopersicum / Q000276:immunology / Q000821:virology*; D008854:Microscopy, Electron; D017830:Octoxynol / Q000737:chemistry; D009994:Osmolar Concentration; D010935:Plant Diseases; D010947:Plants, Toxic; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014026:Nicotiana / Q000276:immunology / Q000821:virology*; D014461:Ultracentrifugation
D000914:Antibodies, Viral; D000956:Antigens, Viral; D005656:Fungal Proteins; D017830:Octoxynol; D006026:Glycoside Hydrolases; C035212:driselase