Two-dimensional gel isoelectric focusing

Miroslava Štastná, Karel Šlais

Research output: Contribution to journalArticle

Abstract

Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90° (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of β-lactoglobulin (pI values 5.14 and 5.31, respectively) nonseparated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.

Original languageEnglish (US)
Pages (from-to)3586-3591
Number of pages6
JournalElectrophoresis
Volume26
Issue number18
DOIs
StatePublished - Sep 2005
Externally publishedYes

Fingerprint

Isoelectric Focusing
Gels
Proton-Motive Force
Lactoglobulins
Glucose Oxidase
Isoelectric Point
Electric potential
Fractionation
Complex Mixtures
Proteomics
Mass spectrometry
Mass Spectrometry
Contamination
Molecular Weight
Molecular weight
Processing

Keywords

  • Gel isoelectric focusing
  • p/ markers
  • Proteins

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Two-dimensional gel isoelectric focusing. / Štastná, Miroslava; Šlais, Karel.

In: Electrophoresis, Vol. 26, No. 18, 09.2005, p. 3586-3591.

Research output: Contribution to journalArticle

Štastná, M & Šlais, K 2005, 'Two-dimensional gel isoelectric focusing', Electrophoresis, vol. 26, no. 18, pp. 3586-3591. https://doi.org/10.1002/elps.200500264
Štastná, Miroslava ; Šlais, Karel. / Two-dimensional gel isoelectric focusing. In: Electrophoresis. 2005 ; Vol. 26, No. 18. pp. 3586-3591.
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