Protein Protocols Protein Protocols

HPSEC 573
84
Molecular Weight Estimation for Native Proteins Using
High-Performance Size-Exclusion Chromatography
G. Brent Irvine
1. Introduction
The chromatographic separation of proteins from small molecules on the basis of
size was first described by Porath and Flodin, who called the process “gel filtration”
(1). Moore applied a similar principle to the separation of polymers on crosslinked
polystyrene gels in organic solvents, but named this “gel permeation chromatography”
(2). Both terms came to be used by manufacturers of such supports for the separation of
proteins, leading to some confusion. The term size-exclusion chromatography is more
descriptive of the principle on which separation is based and has largely replaced the
older names, although the expression “gel filtration” is still commonly used by biochemists
to describe separation of proteins in aqueous mobile phases. Hagel gives a
comprehensive review of the subject with emphasis on proteins (3) and there is also
much useful information in a technical booklet published by a leading manufacturer (4).
Conventional liquid chromatography is carried out on soft gels, with flow controlled
by peristaltic pumps and run times on the order of several hours. These gels are too
compressible for use in high-performance liquid chromatography (HPLC), but
improvements in technology have led to the introduction of new supports with
decreased particle size (5–13 µm) and improved rigidity. This technique is usually
called high-performance size-exclusion chromatography (HPSEC), but is sometimes
referred to as size-exclusion high-performance liquid chromatography (SE-HPLC).
Columns (about 1 × 30 cm) are sold prepacked with the support, and typically have many
thousands of theoretical plates. They can be operated at flow rates of about 1 mL/min,
giving run times of about 12 min, 10–100 times faster than conventional chromatography
on soft gels. A medium pressure high-performance system called fast protein
liquid chromatography (FPLC) that avoids the use of stainless steel components has
been developed by one major supplier (Amersham Pharmacia Biotech). Prepacked
columns of Superose and Superdex developed for this system can also be operated on
ordinary HPLC systems.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
573