| Separation on the basis of size
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| Dialysis and ultrafiltration
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| Figure 2.11 Primary, secondary, tertiary, and quaternary structures. (A) The primary structure is composed of a linear sequence of amino acid residues of proteins. (B) The secondary structure indicates the local spatial arrangement of polypeptide backbone yielding an extended α-helical or β-pleated sheet structure as depicted by the ribbon. Hydrogen bonds between the 'backbone' amide -NH- and -CO- groups stabilize the helix. (C) The tertiary structure illustrates the three-dimensional conformation of a subunit of the protein; while the quaternary structure (D) indicates the assembly of multiple polypeptide chains into an intact, tetrameric protein. |
| Small molecules, such as salts, can be removed from protein solutions by dialysis or ultrafiltration. Dialysis is performed by adding the protein-salt solution to a semipermeable membrane tube (commonly a nitrocellulose or collodion membrane). When the tube is immersed in a dilute buffer solution, small molecules will pass through and large protein molecules will be retained in the tube, depending on the pore size of the dialysis membrane. This procedure is particularly useful for removal of (NH4)2SO4 or other salts during protein purification, since the salts will
interfere with the purification of proteins by ion-exchange chromatography (below). Figure 2.12 illustrates the dialysis of proteins.
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| Gel filtration (molecular sieving)
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| Gel filtration, or gel-permeation, chromatography uses a column of insoluble but highly hydrated polymers such as dextrans, agarose, or polyacrylamide. Gel-filtration chromatography depends on the differential migration of dissolved solutes through gels that have pores of defined sizes. This technique is frequently used for protein purification and for desalting protein solutions. Figure 2.13 describes the principle of gel filtration. There are commercially available gels made from carbohydrate polymer beads designated as dextran (Sephadex series), polyacrylamide (Bio-Gel P series), and agarose (Sepharose series), respectively. The gels vary in pore size, and one can chose the gel filtration materials according to the molecular weight fractionation range desired.
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