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.
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Introduction
Purified proteins serve for different purposes in research and in commercial applications. First of all were proteins purified to study their function, primary and crystal structure. Among the first purified proteins were urease and Concanavalin A.Tradionally were proteins purified using chromatographic methods, or differential precipitation methods such as ethanol precipitation or ammonium sulfate precipitation. However in the age of molecular biology proteins are commonly engineered and they are expressed with a protein tag which allows the use of affinity purification. The protein tag specifically interacts with a affinity resin and binds the tagged protein whereas other proteins are not bound to the resin. This then allows a simple purification by washing the resin with a buffer and release the tagged protein from the resin.
Protein purification for analytical purposes
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. Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low and nanogram amounts of protein are sufficient for their analysis
In the case of a recombinant protein, the yield may be more abundant. Furthermore, purification may be facilitated by the frequent use of a protein tag such as the his-tag- an extra small chain of amino acids at one terminal of the protein which binds specifically to certain resins.
Applications
External link
| Proteins: key methods of study |
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Protein methods | Protein purification | Protein structure prediction | Green fluorescent protein | Western blot | Protein immunostaining | Protein sequencing | Gel electrophoresis | Protein immunoprecipitation | Protein structural alignment | Protein ontology | Enzyme assay | Protein assay | Peptide mass fingerprinting |
