NR AWNJ
AU Schmerr,M.J.; Alpert,A.J.; Yeung,E.S.
TI Chromatographic methods to purify the abnormal prion protein for analysis
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions DIA-44
PT Konferenz-Poster
AB Technologies used for chemical characterization of the proteins require that the protein of interest has a certain level of purity. Current methods used in prion research are ultracentrifugation, sodium phosphotungstic acid precipitation and immunoprecipitation. Although these methods are useful, there are other methods used in analytical chemistry that are more rapid and efficient. In this study, we describe several different chromatographic approaches to purify the abnormal prion protein. Brain from scrapie infected sheep was prepared by grinding the tissue in liquid nitrogen. The finally ground tissue was then resuspended in 0.15M NaCl and extracted with hexafluoro-2-propanol. A phase separation was induced by the addition of 0.5M Na2SO4. The bottom layer which contained the abnormal prion protein was removed and was dried in a vacuum centrifuge. The sample was then resuspended in the appropriate starting mobile phase and centrifuged to remove particulates in preparation for chromatography. Several forms of chromatography were used including normal phase, reverse phase, ion exchange, size exclusion chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography and metal ion affinity chromatography. After chromatography, the peak fractions were tested either by Western blot or by fluorescence immunoassay. One of the major problems that occurred was that the some of the solvents required to solubilize the abnormal prion were incompatible with the column matrices, e.g., - 1% formic acid and 50mM hexafluoro-2-propanol are caustic to some columns. To overcome this problem, small disposable cartridges were substituted for the analytical columns when possible. Application of the chromatographic method adapted to these cartridges was used successfully to develop an assay for sheep blood. These purification methods could be used to differentiate between strains of scrapie.
AD M.J. Schmerr: Ames Laboratory, Iowa State University, Ames, Iowa, USA; A.J. Alpert: PolyLC, Inc. Columbia, Maryland, USA; E.S. Yeung: Ames Laboratory, Iowa State University, Ames, Iowa, USA, and Department of Chemistry, Iowa State University, Ames, Iowa, USA. E-mail: mschmerr@ameslab.gov
SP englisch
PO Italien