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Tris interference in IEF and 2-DE.

Gary B Smejkal1, Myra H Robinson

  • 1Pressure Biosciences, Proteomics and Small Molecules Applications Laboratory, Woburn, MA 01801, USA. gsmejkal@pressurebiosciences.com

Electrophoresis
|April 21, 2007
PubMed
Summary
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Immobilized pH gradient (IPG) hydration with protein solutions reveals Tris accumulation during isoelectric focusing (IEF). This Tris zone causes voltage drops, affecting basic protein migration and potentially hindering acidic protein movement.

Area of Science:

  • Biochemistry and Biophysics
  • Electrophoresis Techniques
  • Proteomics

Background:

  • Dried Immobilized pH Gradients (IPGs) are rehydrated with protein solutions for isoelectric focusing (IEF).
  • Ionic constituents and protein concentrations are initially uniform across the IPG strip.

Purpose of the Study:

  • To investigate the focusing behavior of Tris buffer during IEF in hydrated IPGs.
  • To understand the impact of Tris accumulation on voltage distribution and protein migration patterns.

Main Methods:

  • Isoelectric focusing (IEF) on rehydrated Immobilized pH Gradients (IPGs).
  • Observation of Tris buffer focusing and its interaction with protein migration.
  • Analysis of conductivity and voltage gradients within the IPG strip.

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Main Results:

  • Tris, initially at low concentration, focuses at its pKa, forming a localized zone of high conductivity.
  • This Tris-rich zone causes a sharp voltage drop, influencing the migration of nearby proteins.
  • Basic proteins align at the periphery of the Tris zone, while acidic proteins may be impeded.

Conclusions:

  • Tris buffer's focusing behavior in IPGs significantly impacts electrophoretic conditions.
  • The localized voltage drop can alter protein separation, particularly for basic and acidic proteins.
  • Understanding Tris focusing is crucial for optimizing IEF protocols and interpreting results.