Development of a multilane channel system for nongel-based two-dimensional protein separations using isoelectric focusing and asymmetrical flow field-flow fractionation

Ki Hun Kim, Myeong Hee Moon

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

A. dual purpose multilane channel system to carry out isoelectric focusing (IEF) and asymmetrical flow field-flow fractionation (IEF-AFlFFF or IEF-AF4) was developed for the high-speed fractionation of a proteome in two dimensions (2D): isoelectric point (pI) and hydrodynamic diameter (ds). Separation of proteins is initially achieved by differences in pIusing IEF in an open thin segment, which is formed by interconnecting the beginning part of six parallel flow FFF channels in the lateral direction. After IEF, each protein pool of a different pI interval is simultaneously separated in an orthogonal direction by ds in six individual AF4 channels. The developed IEF-AF4 multilane channel system provides ultimate nongel, elution based, and 2D protein separation at an improved separation speed; the entire separation can be processed within 30 min, compared to ∼3 h with the previously developed capillary isoelectric focusing-hollow fiber FlFFF (CIEF-HFFlFFF or CIEFHF5) (Kang, D.; Moon, M. H. Anal. Chem. 2006, 78, 5789-5798) or ∼36 h with 2D-polyacryamide gel electrophoresis (2D-PAGE). An initial evaluation of IEF-AF4 was performed to investigate the influence of ampholyte concentration and IEF voltage on the separation of standard protein mixtures.

Original languageEnglish
Pages (from-to)1715-1721
Number of pages7
JournalAnalytical Chemistry
Volume81
Issue number4
DOIs
Publication statusPublished - 2009 Feb 15

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Fractionation
Flow fields
Proteins
Isoelectric Focusing
Parallel flow
Moon
Proteome
Electrophoresis
Buffers
Hydrodynamics
Gels
Fibers
Electric potential

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

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