Extended application of gel-permeation chromatography by spin column

Sangeeta Nath, Amrita Brahma, Debasish Bhattacharyya

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Separation of small volumes of proteins from unbound ligands or reequilibration with buffer by passing through a 1-ml Sephadex G-50 column under mild centrifugal force is a popular technique. Here it has been demonstrated that other Sephadex matrix could similarly be used for complete or partial separation of protein molecules. Proteins to be eluted at void volume are recovered near quantitatively, while others are partly or almost completely retained depending on molecular size. Calibration curves using standard proteins of Mw 12.5 to 440kDa with Sephadex G-50-G-200 representing recovery versus molecular weight show profiles as expected from the fractionation ranges of the column matrix. The procedure may be applied to follow protein association-dissociation reactions if the molecular weights of the species concerned are known and a proper matrix exists for separating them. Equilibrium unfolding transitions constructed with model proteins in presence of 0-8M urea using recovery as an index correspond to profiles obtained from other physical measurements. This may be a convenient approach to follow change of protein hydrodynamic volume quickly when a parallel methodology is not readily available.

Original languageEnglish
Pages (from-to)199-206
Number of pages8
JournalAnalytical Biochemistry
Volume320
Issue number2
DOIs
Publication statusPublished - 15-09-2003

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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