Millipore Technical Publications |
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New Workflow for Lentivirus Purification, Concentration, and Immunodetection |
| Lentivirus is a negatively charged, enveloped, single stranded
RNA virus from the Retroviridae family that is often used as a
vector to transport genetic material into cells. These viral vectors
can be used for genetic modification, RNAi, gene therapy, and
vaccine production. Before viral preparations/propagations can be
used for any of these applications, researchers need to purify their
virus sample.
Traditionally, time-consuming density gradient centrifugation separation and/or chromatographic techniques have been used. The lab-scale chromatographic devices are commonly syringe or column-based. To process the virus sample, these devices require hand pressure or gravity; this may lead to messy and to potentially unsafe handling conditions during assembly and disassembly. A new workflow has been developed to clarify, purify, and concentrate/buffer exchange a crude lentivirus sample. For added safety and improved handling, the clarification and purification steps are performed in a closed vacuum-based device. This purification produces high recovery of virus particles in about one hour with similar or improved results as compared to traditional methods. Purity was visualized by gel electrophoresis and confirmed by western blotting using an innovative vacuum-based immunodetection system that allows detection of the protein of interest in less than forty minutes. Here we show the results of the purification and the immunodetection of a Lentivirus-VSVG pseudotype that encodes green fluorescent protein (GFP). We demonstrate the viral titer, the percent recovery of infectious particles, and the purity of the virus sample. Click the PDF above for the full poster. |
