pI Determination of Native Proteins In Biological Samples

Research output: Contribution to journalArticle

Abstract

The electrophoretic mobility of a protein on an immobilized pH-gradient gel (IPG) depends upon its overall positive (acidic) or negative (basic) charge, the principle underlying the IEF technique. In isoelectrofocusing (IEF), a protein with a net positive or negative charge migrates through the pH gradient gel until it reaches the isoelectric point (pI), a pH at which it remains neutral. Thus, the pI of a protein indicates its net charge, a critical determinant of its stability/activity in a given milieu. Conventionally, the first-dimensional IPG-IEF is followed by a second dimension, by which the focused proteins are denatured/reduced and resolved on an SDS-PAGE gel for subsequent immunoblotting to verify the protein identity. The recent development of one-dimensional, vertical IEF followed by immunoblotting enabled concurrent analysis (pI determination) of multiple samples. The protocol described here outlines vertical IEF and immunoblotting under non-denaturing conditions to determine the pI of native proteins in biological samples.

Original languageEnglish (US)
Article numbere85
JournalCurrent Protocols in Protein Science
DOIs
StatePublished - Jan 1 2019

Keywords

  • immunoblotting native protein
  • isoelectric point (pI)
  • isoelectrofocusing (IEF)
  • non-denaturing IEF

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry

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