Protocol

Preparative Size-Exclusion Chromatography

This protocol was adapted from "Size-Exclusion Chromatography," contributed by Helena Hedlund, Chapter 7, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

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INTRODUCTION

Purification of a target molecule in a complex sample and separation of dimers from monomers are two important applications for preparative size-exclusion chromatography (SEC). Choose a column and SEC medium with a selectivity range that gives the best separation of target molecules from contaminants. Column size is chosen based on the volume and concentration of sample. To achieve sufficient resolution for a particular sample volume, use the following method optimization parameters: (a) increase column length by connecting two columns in series; (b) reduce the flow velocity; and (c) reduce the sample volume.

MATERIALS

Reagents

SEC column buffers

Protein sample, extract, or column . . . [Full Text of this Article]

Equipment

METHOD