Posted 19 November 2004 - 11:45 AM
does anyone have a protocol to purify RSV on sucrose ?
This would be very helpful,
Posted 20 November 2004 - 01:58 PM
Posted 30 November 2004 - 11:35 AM
Virus was pelleted by high speed centrifugation at 18000 rpm for 2.5 hours in the SW27 rotor for 20 min at 4°C, then the supernatant was collected and protein concentration was determined by Lowry method.
Posted 30 November 2004 - 11:39 AM
Acta Med Okayama. 1978 Aug;32(4):265-72. Related Articles, Links
Respiratory syncytial virus. I. Concentration and purification of the infectious virus.
Respiratory syncytial (RS) virus can be purified without losing its infectivity provided that each step of purification is carried out using NT buffer containing over 20% sucrose. Firstly, the virus grown on HES cells is efficiently removed from the culture fluid by precipitating with polyethylene glycol (PEG) 6,000, and the precipitate is suspended in a small amount of 20% sucrose-NT buffer, which results in about a 24-fold concentration of the original material. Then this suspension is centrifugated through 30% sucrose-NT buffer to obtain pellets, which are again suspended in 20% sucrose-NT buffer. This suspension is further centrifuged by discontinuous and linear sucrose density gradient. Finally, the specific infectivity of the purified virus was increased about 3,000-fold over that of the original material.