Quick Virus/Viron Purification Protocol
时间:2005-07-18 00:00 来源:Scienceboard.net 作者:admin 点击:次
| Wednesday, October 29, 2003
Description This is a quick method for purifying virus from cell culture without the added hassle of sucrose gradients and identifying fractions positive for virus. This protocol is primarily designed for protein analysis of the collected virions. Procedure Virus containing cell culture media is filtered through a .45 micron filter to remove cells and debris and layered on a 20% sucrose cushion in an Ultracentrafuge tube. Samples are spun at 35,000 rpm for 1-2 hours. Liquid is removed and pellet is resuspended in apropriate buffer (for western analysis SDS page loading buffer). Pipet up and down to resuspend virus. Virus can typically be dissociated by heating at 90C for 15 min. Recipes 20% sucrose (w/v) is made in PBS and then filter sterilized. Store at 4C for several months. Check periodically for contamination. Supplies Ultracentrafuge, with apropriate rotor and tubes etc ex. Sw41, beckman Ultra-Clear 14X89 mm tubes for a 2ml cushion with 9mls of media SDS page buffer, see western protocols. Heat block or boiling water for protein dissociation. Tips Be careful not to mix media with cushion some cellular debris will be in your sample, so good negative controls are needed to ensure specificity of protein to virus Virus can be resuspended in other buffers to concentrate and then use for future infections. (责任编辑：泉水) |