New method for the removal of extraneous proteins from purified oncornaviruses.
Conventional methods (i.e. gradient centrifugation) for the purification of oncornaviruses are usually not effective in complete removal of nonviral proteins. Such contaminants often prove to be a nuisance in subsequent immunological or biochemical studies. Hyperimmune sera prepared from these viruses must be absorbed to assure specificity; cell-derived proteins can be shown to interfere with studies of virus structural proteins, nucleic acids, or viral enzymes. Herein is described a method for removal of most of these contaminants. Viruses are diluted in a high concentration of NaCl to achieve a final concentration of 15%, incubated for 30 min, sedimented, and resuspended in buffer. This procedure results in reductions of up to 48% of the protein without affecting particle count. Immunological, biochemical, and biological properties are not adversely affected. Of the proteins removed, fetal calf serum components and a ribonuclease (presumably cell-derived) were identified. This technique differs significantly from other high-salt methods in that the virus is not precipitated from suspension. It is believed that absorbed proteins are desorbed and left in solution (or suspension) as the virus is sedimented by centrifugation.
D002460:Cell Line; D002499:Centrifugation, Density Gradient; D008722:Methods; D009858:Oncogenic Viruses / Q000201:enzymology / Q000302:isolation & purification* / Q000648:ultrastructure; D011506:Proteins / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011925:Rauscher Virus / Q000302:isolation & purification; D012260:Ribonucleases / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013395:Sucrose; D014764:Viral Proteins / Q000032:analysis
D011506:Proteins; D014764:Viral Proteins; D012965:Sodium Chloride; D013395:Sucrose; D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases