Reversed-phase high-performance liquid chromatography of virus-like particles.
A quantitative reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed to detect the L1 subunit protein from virus-like particles (VLPs) of human papillomavirus (HPV). The method utilizes heat treatment with a buffer consisting of 50 mM Tris, pH 8.0 containing 8 M guanidine-HCl and 10% 2-mercaptoethanol to dissociate the VLPs into monomeric L1. Following dissociation, the sample is injected onto a C4 or C8 column. The L1 protein is eluted as a single, clearly resolved peak. Elution conditions have been optimized to enhance the separation of L1 from other contaminants. Based on spike recovery studies and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis this method is suitable for quantitation of various partially purified in-process samples and can be used to facilitate purification process development.
Yuan
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Y
MedImmune, Inc., Gaithersburg, MD 20878, USA.
Shane
E
E
Oliver
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CN
D015153:Blotting, Western; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007202:Indicators and Reagents; D027383:Papillomaviridae / Q000302:isolation & purification*; D012997:Solvents; D014764:Viral Proteins / Q000302:isolation & purification; D014780:Viruses / Q000302:isolation & purification*
D007202:Indicators and Reagents; D012997:Solvents; D014764:Viral Proteins