Biochemistry: Protein separation and purification

Purification and separation of biochemical proteins gel electrophoresis; SDS - PAGE. Salting and salting out; dialysis. Gel filtration (ie size exclusion) chromatography. Ion exchange chromatography

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Protein purification is essential for characterizing the function, structure and interaction of proteins. Each step of the purification process may involve cell lysis, separation of soluble protein components from cellular debris, and ultimately separation of the protein of interest from product and process related impurities. Separating the target protein from all impurities in the desired form is often the most difficult aspect of protein purification. Affinity chromatography is an efficient protein purification technique that typically enhances one step purification of a protein to a level of purity sufficient for analytical characterization. Affinity chromatography is a molecular conformation based separation technique in which molecules "lock" and key "modes selectively bind to each other" (eg, antibodies recognize and specifically bind antigens).

Affinity chromatography is based on selective noncovalent interactions between analytes and specific molecules. It is very special, but not very powerful. It is commonly used in the purification of proteins bound to labels in biochemistry. These fusion proteins are labeled with compounds such as His tag, biotin, or antigen that bind specifically at stationary phase. After purification, some of these tags are typically removed and pure proteins are obtained. However, there is HPLC technology utilizing affinity chromatography characteristics. Immobilized metal affinity chromatography (IMAC) can be used to separate these molecules based on their relative affinity for metals (ie Dionex IMAC). Typically, these columns can be filled with different metals to create columns with target affinity.

Affinity chromatography based on selective noncovalent interactions between analytes and specific molecules is very specific, but less robust. It is generally more commonly used in biochemistry to purify proteins bound to tags. Affinity chromatography typically utilizes the affinity of biomolecules for metals (Zn, Cu, Fe, etc.). Columns are usually prepared manually. Conventional affinity columns are used as a preparation step to wash unwanted biomolecules. Ion exchange chromatography, also known as ion chromatography, uses an ion exchange mechanism to separate analytes according to their respective charges. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. Ion exchange chromatography is commonly used to purify proteins using FPLC