Please find the following protocol:

Purification of Rous Sarcoma Virus (RSV)


Procedure:

1. Spin down virally-infected cells and debris for 10 min at 8,000
rpm (8,000 X g) in an SS-34 rotor at 4°C. Save the
supernatant. The virus can be kept frozen at -70°C after it has been clarified. It should not be frozen before it has been clarified, as this disrupts the floating cells and causes an increase in the amount of debris. The virus is stable to freeze-thawing in medium, but not after it has been purified, unless Serum or Bovine Serum Albumin is added back. The virus should be kept on ice or at 4°C during purification; it should not be frozen until the purification is complete.

2. Concentrate the supernatant if necessary. Slowly, and with
stirring, add an equal volume of saturated Ammonium Sulfate to the supernatant. Incubate the sample on ice for 15 min. Centrifuge for 10 min in an SS-34 rotor at 16,000 rpm (30,000 X g) at 4°C. Pour off the supernatant, and remove any remaining drops with a Kimwipe. Traces of Ammonium Sulfate will make it difficult to redissolve the pellet. Resuspend the virus pellet in SB to 5% of the starting volume. The suspension should be clear; if not add more SB and/or reclarify by repeating Step #1.

3. Prepare a Sucrose step gradient that consists of a cushion of 65% Sucrose in SB, a layer of 20% Sucrose in SB, and the virus suspension. The solutions can be overlayed successively, starting with 65% cushion, or underlying successively, starting with the virus suspension.

4. Sediment the virus using the appropriate speeds and times below. The longer the centrifugation time the greater the contamination by slower-sedimenting particles (membrane vesicles, etc.) in the virus suspension.

Approximate volumes and centrifuge times:

Rotor / Tube / Volume / 65% Sucrose / 20% Sucrose / Virus / Suspension / rpm / Time

SW50.1 / 0.5" x 2" / 5 ml / 0.5 ml / 2 ml / 2 ml / 50,000 / 30 min

SW41 / 0.56" x 3.5" / 13 ml / 0.7 ml / 6 ml / 6 ml / 41,000 / 45 min

SW27 / 1" x 3.5" / 38.5 ml / 1 ml / 18 ml / 18 ml / 27,000 / 60 min

SW25.1 / 1" x 3" / 34 ml / 1 ml / 16 ml / 16 ml / 25,000 / 60 min

5.Collect the interface between the 20% and 65% Sucrose Solutions.

6. Dilute the virus 1:4 with SB so that the density is less than that of the 20% Sucrose. In order to get a purer preparation, the virus can be sedimented through 20% Sucrose (i.e. repeat Step #3 and #4) after the material at the 20%/65% interface has been diluted with SB. In addition, small amounts of crude medium or concentrated virus could be layered onto a 5 to 20% Sucrose gradient and a velocity separation carried out (e.g. 10 min at 30,000 rpm in the SW41).

7. Load the virus onto a 20% to 55% Sucrose gradient.

8. Centrifuge the viral solution for twice the time centrifuged in Step #4 using the same rotor. The virus will band at a density of approximately 1.16 (37% Sucrose). Since this is an equilibrium separation, the centrifuge can be run for a longer time if necessary.

9. Collect the gradient and determine the density of each fraction, by weighing 100 μl in a Lang-Levy micropipet or by determining the refractive index.

10. Collect the purified virus.




Standard Buffer (SB)*........................................0.001 M EDTA
............................................0.01 M Tris
.....................................Adjust pH to 7.4
..........................................Store at 4°C
.............................................0.1 M NaCl

Saturated Ammonium Sulfate Solution
.............................................2 ml 0.5% (w/v) Phenol Red
............................................................531 g (NH4)2SO4
Bring up to 1 liter with ddH2O, neutralize the pH with Ammonia Store at room temperature.

Sucrose Solutions in SB20,
% Sucrose (w/v), Density, Refractive Index
20, 1.08, 1.3639
55, 1.26, 1.4307
65, 1.32, 1.4532
Store Sterile at 4°C


* Smith and Bernstein (see Citation #2) state that this Buffer, without 0.1 M NaCl, gives less virus aggregation and purer virus preparations.


BioReagents and Chemicals

Sucrose
Ammonia
Phenol Red
Ammonium Sulfate
Tris
EDTA
Sodium Chloride
Bovine Serum Albumin



Reference and Links:

http://www.bio.com/protocolstools/protocol.jhtml?id=p301

1. Duesberg PH, Robinson HL, Robinson WS, Heubner RJ, Turner HC. Proteins of Rous sarcoma virus. Virology 1968; 36:73

2. Smith RC, Bernstein EH. Production and purification of large amounts of Rous sarcoma virus. Appl. Microbiol. 1973; 25:346