Protein purification
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Protein purification is the process of isolating proteins from a homogenate, which may comprise cell and tissue components, including DNA, cell membrane and other proteins.
Purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isolectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Finally, proteins may be isolated according to their unique binding properties. For example, Lactate Dehydrogenase (LDH), which binds NADH, can be purified by running it through a column comprised of beads with prosthetic NAD analog groups.
Protein purification schemes usually involve multiple separations and isolations. Furthermore, two purification methods may be employed together to increase the degree of purification. For example, in two dimensional gel electrophoresis, proteins are first separated longitudinally according to molecular weight, then laterally according to their isoelectric points.
If the protein comes from a natural source (e.g. plant or animal tissues), it is often necessary to process a large amount of the source material to isolate a small amount of the target protein (typically, milligram or sub-milligram level).
In the case of a recombinant protein, the yield may be more abundant. Furthermore, purification may be facilitated by the frequent use of a recombinant tag - an extra small chain of amino acids at one terminal of the protein which binds specifically to certain resins.
External link
- SDS-PAGE: Methods and Theory (http://www.westernblotting.org/protocols%202.html)