Thin-Layer Gel Filtration

Determination of Protein Molecular Weights
  • John M. Walker
Part of the Springer Protocols Handbooks book series (SPH)


For many years Sephadex gel filtration of proteins has been a valuable tool for separating proteins on the basis of size difference. Sephadex is a bead-formed gel prepared by crosslinking dextran, and is available in a variety of G-types that differ in their degree of crosslinking (1). Depending on the G-type used, proteins in various molecular weight ranges can be fractionated. The basic principle of this separation method is as follows. A column is packed with Sephadex beads and buffer is pumped through to equilibrate the column. A protein mixture is then pumped onto the column, and as the sample passes through the column the individual protein components begin to separate according to their sizes. Essentially, small protein molecules, which can effectively penetrate the pores of the Sephadex beads, flow more slowly down the column than high-molecular-weight proteins that are too large to penetrate any of the beads. Proteins of intermediate size that can enter some beads, but not others (the beads are not all of identical pore size), are correspondingly retarded to a greater or lesser extent.


Elution Volume Molecular Weight Range Buffer Reservoir Small Protein Molecule Sephadex Bead 
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  1. 1.
    Gel Filtration—Theory and Practice. A free booklet provided by Pharmacia Fine Chemicals AB, Uppsala, Sweden.Google Scholar
  2. 2.
    Andrews, P. (1971) Estimation of Molecular Size and Molecular Weight of Biological Compounds by Gel Filtration, in Methods of Biochemical Analysis Vol. 18, Glick, D., ed. Interscience, New York.Google Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • John M. Walker
    • 1
  1. 1.Division of Biological and Environmental StudiesThe Hatfield PolytechnicUK

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