|
| 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 |
|
