Updated - Copyright 2014 Molecular Station
Protein purification is vital in the characterization of your protein of interest. Purification of your protein allows one to study the function of the protein, and its enzymatic activity. Stuctural information from the protein can also be obtained from purified proteins including NMR, 3-D information such as protein crystallization.
So how does one go about purifying a protein?
Proteins can be purified according to size, solubility, Charge and Binding affinity
Proteins can readily be visualized and differentiated by electrophoresis methods. These gel techniques can also be used to obtain small quantities (micrograms) of purified polypeptides. However, they do not provide large amounts of purified proteins in their native state. Substantial quantities of purified proteins, of the order of many milligrams, are needed to elucidate fully their three-dimensional structure and their mechanism of action. Several thousand proteins have been purified in active form on the basis of such characteristics as size, solubility, charge and specific binding affinity. At each step in purification, the preparation is assayed for a distinctive property of the protein of interest (e.g. enzymatic activity) to assess the efficacy of the procedure.
Proteins can be separated from small molecules by dialysis through a semi-permeable membrane, such as cellulose membrane with pores. Molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis bag, whereas smaller molecules and ions traverse the pores of such a membrane and emerge in the dialysis outside the bag.
More discriminating separation on the basis of size can be achieved by the technique of gel-filtration chromatography. The sample is applied to the top of the column consisting of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose (which are carbohydrates) or polyacrylamide. Sephadex, Sepharose, and Bio-gel are commonly used commercial preparations of these beads, which are typically 100 micrometers in diameter. Small molecules can enter these beads but large ones cannot. The result is that small molecules are distributed both in the aqueous solution inside the beads and between them, whereas large molecules are located only in the solution between the beads. Large molecules flow more rapidly through this column and emerge first, because a smaller volume is accessible to them. It should be noted that the order of emergence of molecules from a column of porous beads is the reverse of the order in gel electrophoresis, in which a continuous polymer framework impedes the movement of large molecules. Much larger quantities of protein can be separated by gel filtration chromatography than by gel electrophoresis but at the price of lower resolution.
The solubility of most proteins is lowered at high salt concentrations. This effect, called salting out, is very useful, though not well understood. The dependence of solubility on salt concentration differs from one protein to another. Hence salting out can be used to fractionate proteins. Salting out is also useful for concentrating dilute solutions of proteins.