Evaluation of non-immunoaffinity methods for isolation of cellular prion protein from bovine brain.
Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that affect the central nervous system of many animals, including humans. Research suggests that TSEs are caused by conversion of the cellular prion protein (PrP(C)), which is encoded in many tissues, especially brain, to the pathological form (PrP(Sc)). This conversion affects PrP(Sc) structure, conferring different biochemical properties, such as the increased resistance to proteinase K, that have been widely used for its purification. By contrast, PrP(C) is less resistant and its isolation is more challenging. Here, we propose a purification strategy to efficiently recover PrP(C) from healthy bovine brain using conventional non-immunoaffinity methods. The applicability of extraction using detergents, size exclusion chromatography, diafiltration with molecular weight cutoff (MWCO) filters, and immobilized metal affinity chromatography (IMAC) using Western blot (WB) analysis to detect the presence of PrP(C) is discussed in detail.
Department of Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain.
M
M
Borges-Alvarez
Department of Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain. Electronic address: fbenavente@ub.edu.
F
F
Benavente
Animal Tissue Bank of Catalunya, Department of Animal Medicine and Surgery, Autonomous University of Barcelona, 08193 Bellaterra, Spain.
M
M
Márquez
Department of Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain.
J
J
Barbosa
Department of Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain.
V
V
Sanz-Nebot
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D018621:PrPC Proteins / Q000032:analysis / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014462:Ultrafiltration
D018621:PrPC Proteins
Cellular prion protein; IMAC; Purification; SEC; Western blot