|
Isolation and purification steps | Authors |
Geering and Thomas, 1999 | Hammond et al., 2006b | Mukhamedzhanova et al., 2011; Ivanov et al., 2011 | Donchenko et al., 2017 |
|
Yield (mg of virus/100 g of green plant biomass) and host plants used | 23.4 /Chenopodium amaranticolor/ | 8.6-12.4/Nicotiana benthamiana/ | 3.4 /Portulaca grandiflora/ | 20.0 /Portulaca grandiflora/ 57.3/Nicotiana benthamiana/ |
|
Homogenization buffer | 0.02 М Sodium borate buffer, 0.5% Na2SO3, рН 8.2 (250 ml buffer per 150 g of leaves) | 0.5 M K2H/KH2P04, 0.5% Na2SO3, рH 8.4 (3-5 buffer volumes per weight of leaves) | 0.3 М glycine-КОН, 1% Na2SO3, рН 7.5 (3 ml buffer per 1 g of leaves) | 0.3 М glycine-КОН, 1% Na2SO3, рН 7.5 (3 ml buffer per 1 g of leaves) |
|
Virus enrichment from plant tissue | centrifugation 0.5% Triton X-100 | centrifugation 2% Triton X-100, 4% PEG 8000, 2% NaCl | centrifugation 1% Triton X-100 5% PEG 6000, 2% NaCl (Personal communication) | centrifugation 1% Triton X-100 Two stages of precipitation: (1) 5% PEG 6000, 2% NaCl (2) 8% PEG 6000 |
|
Extraction of virus from pellets | — | Extraction in 0.1 М Sodium borate buffer, 0.1 М KCl, рН 8.0 (BK buffer), 0.75-1.5 hours. | Extraction in 0.05 M Tris-HCl, 0.01 M EDTA, pH 8.0, (Personal communication) | Extraction in 0.05 М Tris-HCl, 0.01 М EDTA, рН 8.0, Two stages for 2-6 hours. |
|
Ultracentrifugation steps | (1) Separation by 10-40% sucrose gradient (0.01 М Tris-HCl, 0.001 М EDTA, рН 8.0) (85 000 g, 4 hours) (2) Virus precipitation (85 000 g, 2.5 hours) | (1) Separation by 30% sucrose cushion (BK buffer) (85 600 g, 2.5 hours) (2) Separation by CsCl gradient, density 1.32 g/cm3 (139 000 g, 16-20 hours) (3) Dialysis against 0.5х BK buffer in 1 l (3 changes) | (1) Virus precipitation (100 000 g, 1.5 hours) (Personal communication) (2) Separation by 30% sucrose cushion (110 000 g, 2.5 hours) (Personal communication) | (1) Virus precipitation (111 000 g, 3 hours) (2) Separation by 30% sucrose cushion (extraction buffer) (111 000 g, 4 hours) |
|
Spectrophotometric analysis (,0.1) | 2.84 | 2.5 | 2.84 | 2.84 |
|