The Isolation of Friend Virus Protein by Zonal Centrifugation
Gilbreath, Michael Joye
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The problem. Isolation of Friend virus protein in an increased yield for immunological purposes by zonal centrifugation was the purpose of this experiment. Procedure. Sucrose gradient zonal centrifugation was used to isolate Friend virus polysomes. The concentration of the polysomes and the removal of the sucrose was accomplished by ultrafiltration. Puromycin was used to release the nascent viral protein from the polysomes isolated, and the nascant protein was separated from the polysomes by centrifugation and then concentrated using ultrafiltration. Findings. 1480 mg of protein was isolated from 30 Friend virus infected mice spleens. This amount was significantly more than the amount expected. Aggregates of soluble protein are believed to have sedimented along with polysomes and these proteins may contaminate the Friend virus protein isolated. Only partial release of the nascent protein was obtained in this study. Conclusion. The use of sucrose as a separating medium does not separate large aggregates of soluble protein from the polysomes during zonal centrifugation. The use of puromycin in large amounts to release a usable quantity of nascent protein may make this method impractical due to high cost. Recommendations. Cesium chloride should be used in place of sucrose in zonal centrifugation in an effort to separate the aggregates of soluble protein from the polysomes. An attempt to increase the release of nascent protein with puromycin should be made. The isolated protein should be innoculated into BALB/c mice to ascertain the effectiveness of the viral protein as an immunizing agent and the possible deleterious effect of the contaminating soluble protein.
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