Dynamics of gel isoelectric focusing with ampholytic dyes monitored by camera in real-time

Miroslava Štastná, Karel Šlais

Research output: Contribution to journalArticlepeer-review

Abstract

The dynamics of gel isoelectric focusing were studied by using amphoteric low-molecular-mass colored substances (isoelectric point markers). The polyacrylamide gel in slab format was in direct contact with the electrodes. In addition to isoelectric focusing with a pH gradient composed of synthetic carrier ampholytes, pH gradients created by simple buffers of acetic acid, 2-(N-morpholino)ethanesulfonic acid, histidine and N,N,N′,N′-tetramethylethylenediamine were applied. The progress of the electrofocusing process was monitored by a charge-coupled device camera and video recording. The gradient profile and dynamics were approximated from the positions of isoelectric point markers, which were focused both on boundaries between individual zones of simple buffers and within the zones themselves. The obtained animated records enabled the observation of the entire real focusing run within fractions of a minute, which is useful both for the understanding and optimization of the focusing.

Original languageEnglish (US)
Pages (from-to)193-203
Number of pages11
JournalJournal of Chromatography A
Volume1008
Issue number2
DOIs
StatePublished - Aug 8 2003
Externally publishedYes

Keywords

  • Detection, electrophoresis
  • Dyes
  • Isoelectric focussing
  • Isoelectric point markers
  • Polyacrylamide gels
  • Video monitoring

ASJC Scopus subject areas

  • Analytical Chemistry

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