DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 15

There are a number of ion exchange resins commonly used includ-
ing anionic cellulose or dextran resins like DEAE-cellulose or DEAE-
Sephadex, respectively, and cationic resins including CM-cellulose and
CM-Sephadex.
Adsorption and partitioning matrices
Adsorption and portioning matrices retard the flow of a protein based
on its physical characteristics. There are a large number of gels that func-
tion in this capacity. One example is hydrophobic interaction chromatog-
raphy which is a method used to enrich proteins that have significant
hydrophobic groups. These columns generally are hydrophilic in nature.
Most columns used in hydrophobic chromatography include a phenyl
agarose matrix system. Common matrices include Phenyl-Sepharose
and octyl-sepharose gels.
Affinity chromatography
Affinity chromatography is a method to specifically isolate a protein
based on one of two methods. The most common form of affinity chro-
matography is an antibody based method in which antibodies that are
specific for a particular protein are covalently coupled to a column matrix,
often some form of Sepharose activated by an agent such as cyanogen-
bromide. Material containing the antigen of interest, such as extracts
of cells in which recombinant proteins have been expressed or super-
natants from cultures in which proteins have been secreted during tissue
culture, are passed over the columns. Only those proteins for which the
antibodies are specific will bind to these columns. After washing, they
can then be eluted with at low pH (2.5-3.5) or using chaotropic salts.
Chaotropic salts (such as sodium thiocyanate or ammonium sulfate),
like low pH, disrupt antigen-antibody and protein-protein interactions,
allowing the bound material on the column to be eluted. Affinity chro-
matography is frequently used in purifying recombinant proteins be-
cause it gives one of the highest degrees of purity in a single step of any
chromatographic method.
In addition to the use of antibodies, other agents are used for affinity
chromatography. For example stretches of 6 histidine residues (intro-
duced by recombinant DNA technology at the N or C terminus of a
protein) bind to nickel. Therefore, nickel columns are used to purify his-
tagged recombinant proteins. Protein A and protein G are frequently
used to purify antibodies (see Chapter 4) In addition, lectins, which are
proteins that recognize specific carbohydrate moieties, are frequently
used to purify giycoproteins by affinity chromatography. Lectins can also