DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 14

designed to trap or retard the passing of macromolecules based on a
particular property such as size, charge, or composition. It is commonly
used in protein purification. The mixture to be separated is added to the
column and is flushed through by adding volumes of a buffer designed
for the column. Flow is achieved by gravity or small pumps, although
specialized equipment can also be used (see HPLC and FPLC below).
Material flowing through the column is collected in tubes by automated
fraction collectors for further analysis. The most common forms of chro-
matography are summarized below.
Gel filtration (or “size exclusion”) chromatography
Gel filtration chromatography is based on a sieving method in which
proteins are separated on the base of their size. Usually, columns are
packed with a porous gel. The pores in the gel, which are essentially
small holes, allow molecules of a predefined size or smaller to enter
the pores, whereas the larger molecules are excluded. These larger
molecules flow quickly through the gel and exit the column first, while
the smaller molecules flow more slowly. Therefore, larger volumes of
liquid are required to flush the smaller molecules out of the gel. In gen-
eral, the volume required is inversely proportional to the size of the
molecule. Unlike other methods of column chromatography, gel filtration
chromatography is a method in which all proteins are expected to flow
through the column and are separated based on the speed at which
they exit. All other chromatographic techniques are based on retention
of proteins by one or more properties.
Common media for gel filtration columns include agarose-based
beads, such as Sepharose and Superose, cellulose based beads, and
composites (agarose and dextran = Superdex; dextran and acrylamide
beads = Sephacryl).
Ion exchange chromatography
Ion exchange chromatography is a method used to isolate proteins
based on their charge. Both anion exchange columns (positively charged
columns that are used to isolate negatively charged proteins) and cation
exchange columns (negatively charged columns to isolate positively
charged molecules) are available. Proteins that are bound to ion ex-
change columns are generally released or “desorbed” by increasing the
salt concentration, which is often accomplished using a salt gradient.
The higher the charge of the protein, the higher the salt concentration
required to desorb it. Alternatively, altering the pH of the buffers can be
used to desorb the bound proteins.