RESUMO
The formation of ß-sheet-rich prion protein (PrP(ß)) oligomers from native or cellular PrP(c) is thought to be a key step in the development of prion diseases. To assist in this characterization process we have developed a rapid and remarkably high resolution gel electrophoresis technique called RENAGE (resolution-enhanced native acidic gel electrophoresis) for separating, sizing, and quantifying oligomeric PrP(ß) complexes. PrP(ß) oligomers formed via either urea/salt or acid conversion can be resolved by RENAGE into a clear set of oligomeric bands differing by just one subunit. Calibration of the size of the PrP(ß) oligomer bands was made possible with a cross-linked mouse PrP(90-232) ladder (1- to 11-mer) generated using ruthenium bipyridyl-based photoinduced cross-linking of unmodified proteins (PICUP). This PrP PICUP ladder allowed the size and abundance of PrP(ß) oligomers formed from urea/salt and acid conversion to be determined. This distribution consists of 7-, 8-, 9-, 10-, and 11-mers, with the most abundant species being the 8-mer. The high-resolution separation afforded by RENAGE has allowed us to investigate distinctive size and population changes in PrP(ß) oligomers formed under various conversion conditions, with various construct lengths, from various species or in the presence of anti-prion compounds.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Príons/análise , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Animais , Calibragem , Eletroforese em Gel de Poliacrilamida/normas , Luz , Camundongos , Compostos Organometálicos/química , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Príons/normas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sais/química , Ureia/químicaRESUMO
BACKGROUND AND OBJECTIVES: A standard panel of materials is needed for the evaluation of assays being developed for the diagnosis of variant Creutzfeldt-Jakob disease. MATERIALS AND METHODS: Tissues from human and animals incubating transmissible spongiform encephalopathy disease have been prepared, aliquoted and where possible characterized by in vitro methods. RESULTS: A standardized preparation of materials has been generated. CONCLUSIONS: Large-scale preparations of tissues and blood fractions can be used to directly compare the sensitivities of assays using different formats.