An aqueous two-phase system as a strategy for serum albumin depletion.
An aqueous two-phase system, based on the cloud point phenomenon, was proposed for blood plasma albumin depletion prior to gel electrophoresis (SDS-PAGE) analysis. Albumin removal was evaluated using a mixture of Triton X-114 and SDS. Variables affecting phase separation and the partition coefficient (K), such as pH (from 5.0 to 7.6) and salt composition, were first optimized to carry out phase separation at room temperature and to promote albumin removal (15 min with ca. 95% efficiency) to the surfactant-rich phase, in a global process that results in reduction of 12% alpha-helix structures, confirmed through circular dichroism analysis. Gel electrophoresis analyses were carried out to compare crude human plasma composition with human plasma submitted to the cloud point procedure as well as the same plasma submitted to the separation of albumin using a commercially available system based on a chromatographic affinity column. The cloud point extraction procedure allowed identification of 18 protein bands present in the surfactant-poor phase, resulting in a more detailed electrophoretic profile, when compared with crude plasma (10 protein bands).
da Silva
Marcelo Anselmo Oseas
MA
Department of Analytical Chemistry, Group of Spectrometry, Sample Preparation and Mechanization, Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.
Arruda
Marco Aurélio Zezzi
MA
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004573:Electrolytes; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D017830:Octoxynol; D011092:Polyethylene Glycols; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D013696:Temperature; D013997:Time Factors
D004573:Electrolytes; D008823:Micelles; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40