title: Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers.
abstract: The formation of β-sheet-rich prion protein (PrP(β)) 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(β) complexes. PrP(β) 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(β) 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(β) 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(β) oligomers formed under various conversion conditions, with various construct lengths, from various species or in the presence of anti-prion compounds.
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E8.
authors.forename: Carol L
authors.initials: CL
authors.lastname: Ladner
authors.forename: David S
authors.initials: DS
authors.lastname: Wishart
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D002138:Calibration; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards; D008027:Light; D051379:Mice; D009942:Organometallic Compounds / Q000737:chemistry; D018621:PrPC Proteins / Q000235:genetics / Q000378:metabolism; D011328:Prions / Q000032:analysis* / Q000592:standards; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D009942:Organometallic Compounds; D018621:PrPC Proteins; D011328:Prions; D011994:Recombinant Proteins; D012492:Salts; C103599:bis(bipyridyl)ruthenium(II); D015082:2,2'-Dipyridyl; D014508:Urea