Chemicals Used in Chromatography

In addition to buffers and salts, the mobile phases used for protein chromatography often include a variety of other additives.

Chemical Class Function/Application
TPCK, TLCK Protease Inhibitor Proteases such as trypsin and chymotrypsin are ubiquitous in eukaryotic cell extracts. These inhibitors (tosyl-L-lysine chloromethyl ketone or TLCK for trypsin, tosyl-L-phenylalanine chloromethyl ketone or TPCK for chymotrypsin) prevent the proteases from digesting the protein before it is purified
Glycerol, maltose, trehalose Stabilizing sugars These sugars have been found to extend the shelf-life of many unstable proteins. Glycerol is often used as an additive for plant proteins.
EDTA Chelator EDTA binds heavy metals, such as lead or mercury, that might inactivate proteins. EDTA must be used with care, since it can also chelate metal ions that are required for enzyme activity.
2-mercaptoethanol, dithiothreitol Reduced thiol reagents These compounds are added to prevent oxidation of thiol groups on cysteine side chains, which are often necessary for structure and function.
Antioxidant In enzymes that are sensitive to air oxidation, buffers are often sparged with one of these gases to drive the oxygen out of the solutions. This is very good precaution to use for almost any pure or nearly pure proteins.

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