Molecular Weight Estimation for Native Proteins Using High-Performance Size-Exclusion Chromatography

Le résumé fourni par la source

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. Comprehensive reviews that cover both practical and theoretical aspects of the technique are available, with emphasis on both proteins (3, 4) and peptides (5), and there is also much useful information in a technical booklet published by a leading manufacturer (6). 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 chromatog-raphy (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 chromato-graphy (FPLC) that avoids the use of stainless steel components was developed by one major supplier (Amersham Pharmacia Biotech, now GE Healthcare Life Sciences). Prepacked columns of Superose and Superdex developed for this system can also be operated on ordinary HPLC systems. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.