Striving for Purity: Advances in Protein Purification

With the human genome sequenced, increasing numbers of researchers are turning their attention to proteins—with the hurdle that they must be purified first. A pure sample of protein is required to generate antibodies, conduct binding assays and study structure. Yet the target protein must first be isolated; the debris, salts and reagents washed away; the amount of protein quantified; and, often, the sample concentrated. A major challenge, according to Joern Kirchhuebel, project manager of Proteome Analysis at Eppendorf, is to retain proteins’ native conformations while bringing them into solution. Yet the difficulties, and the importance, of protein work cannot be overstated when it comes to understanding the mechanisms of many, if not all, physiological pathways. Studying the complexities of protein-protein interactions is no easy feat, yet it is vital to unraveling the mysteries of conditions such as Alzheimer disease, diabetes and cancer.

Source from: http://www.nature.com/nmeth/journal/v2/n1/full/nmeth0105-71.html

Strategies for Protein Purification

Strategies for Protein Purification – Soluble Extracellular Proteins

The source of soluble extracellular proteins is the extracellular medium, whether it could be an animal source such as blood or spinal fluid, or a culture medium in which bacterial, fungal, animal, or plant cultures have been grown. Generally these do not contain a large number of different proteins (blood is an exception), and the desired protein may be a major component, especially if produced as the result of recombinant expression. Nonetheless, the protein in the starting material may be quite dilute, and a large volume may therefore need to be processed. The starting fluid may also contain many compounds other than proteins, whose behavior must be taken into account. The first stage should aim mainly to reduce the volume and get rid of as much nonprotein material as possible; some protein-protein separation is also useful, but not essential. No general rules can be given, but a batch adsorption method using an inexpensive material such as hydroxyapatite, ion-exchange resin, immobilized metal affinity chromatography (IMAC) medium, or affinity adsorbent is best, if feasible. Following the first step, the sample should be in a form that is amenable to standard purification processes such as precipitation and column chromatography.

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Protein Purification Methods Based on Surface Features

Surface features include charge distribution and accessibility, surface distribution of hydrophobic amino acid side chains, and, to a lesser extent, net charge at a given pH (see discussion of net charge). Methods exploiting surface features mainly depend on solubility properties. Differences in solubility result in precipitation by various manipulations of the solvent in which the proteins are solubilized. Methods for obtaining an extract containing the desired protein in soluble form are given in another chapter. The solvent, nearly always water containing a low concentration of buffer salts, can be treated to alter properties such as ionic strength, dielectric constant, pH, temperature, and detergent content, any of which may selectively precipitate some of the proteins present. Conversely, proteins may be selectively solubilized from an insoluble state by manipulation of the solvent composition. The surface distribution of hydrophobic residues is an important determinant of solubility properties; it is also exploited in hydrophobic chromatography, both in the reversed phase mode and in aqueousphase hydrophobic-interaction chromatography.

Reprinted from http://www.recombinant-protein.com/protein-purification-methods-based-on-surface-features/