Grow Nicotiana benthamiana plants for 6–8 weeks.

Inoculate each leaf by sprinkling a small amount of carborundum over the plants then gently rubbing 100 μL of 0.01 mg/mL TMV in KP buffer per leaf (seeNote 7).

Collect infected leaves 7–10 days post-infection when mosaic patterns present themselves and before the leaves die.

Immediately freeze leaves and store in Ziploc bags at −80 °C (seeNote 8).

When ready, pulverize frozen leaves by squeezing bag (seeNote 9).

Homogenize leaves in 3 volume of prechilled (4 °C) 0.1 M KP buffer (~300 mL), pH 7.4 and 0.2 % (v/v) β-mercaptoethanol (~600 μL).

Filter homogenate through 2 layers of cheesecloth; squeeze cheesecloth to collect all of the filtrate.

Centrifuge the filtrate for 20 min at 11,000 × g (10,500 rpm when using Beckman Coulter JLA-16.250 rotor).

Carefully pour the supernatant through 4 layers of Kimwipes.

To the supernatant (approximately 300 mL) add equal volume of 1:1 chloroform:n-butanol (150 and 150 mL).

Stir for 30 min on ice (avoid foaming by controlling the stirring speed).

Centrifuge for 10 min at 4,500 × g (6,000 rpm when using Beckman Coulter JLA-10.500 rotor).

Carefully collect the aqueous phase (~300 mL, top layer that contains the TMV) using a pipette and store on ice (seeNotes 10 and 11).

Add NaCl to 0.2 M (3.5 g), PEG 8 k to 8 % (w/v) (24 g), Triton-X 100 surfactant to 1 % (v/v) (3 mL) to the collected supernatant and put the mix on ice.

Stir on ice for 30 min, then store in refrigerator for at least 1 h.

Centrifuge for 15 min at 22,000 × g or max speed of centrifuge/rotor) (15,000 rpm when using Beckman CoulterJLA-16.250 rotor) (seeNote 12).

Resuspend the pellet in 15 mL 0.1 M phosphate buffer (~0.05 mL/g leaf) by carefully pipetting up and down or by incubating on a shaker for 4 h to overnight at 4 °C (seeNote 13).

Centrifuge for 15 min at 9,000 × g (9,500 rpm when using Beckman Coulter JLA-16.250 rotor).

Layer the supernatant on a sucrose gradient (see Subheading 2) and centrifuge in a swing bucket rotor for 2 h at 96,000 × g (28,000 rpm when using Beckman Coulter SW 32 Ti rotor).

Collect the light scattering region (seeFig. 3) and dilute with 0.01 M phosphate buffer to fill ultracentrifuge tube.

Centrifuge in a fixed angle rotor for 2.5 h at 160,000 × g (42,000 rpm when using Beckman Coulter type 50.2 Ti rotor).

Discard supernatant and resuspend pellet in 0.01 M KP buffer overnight.

Centrifuge the resuspended pellet for 15 min at 7,500 × g (10,000 rpm when using Beckman Coulter FX241.5P rotor) in a table top centrifuge (e.g., Beckman Coulter Microfuge 16) and save supernatant: pure TMV solution.

Test concentration using UV–Vis absorbance (e.g., Nanodrop). TMV has an A260 nm = 0.3 for a 1 mg/mL solution and 1 cm path length. An A260/A280 ratio equal to 1.2 indicates intact TMV particles.

Test purity using SEC with a Superose 6 10/300GL column by monitoring the absorbance at 260 nm.