title: Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis.
abstract: A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.
authors.affiliation: Division of Industrial Food Research, National Food Institute (DTU Food), Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark. Electronic address: fjes@food.dtu.dk.
authors.forename: Flemming
authors.initials: F
authors.lastname: Jessen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain), Kogle Alle 6, DK-2970 Hørsholm, Denmark.
authors.forename: Tune
authors.initials: T
authors.lastname: Wulff
mesh_terms: D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D001798:Blood Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40
keywords: 2D electrophoresis; Cloud point extraction; Lipoproteins; Prefractionation; Serum amyloid A; TX-114