Which techniques for purification of proteins can be made specifically for a given protein?

In biochemistry, it is important to control purification processes so that analyses of proteins can isolate the charactersitics of each protein separately. That protein to be examined may be only 1% of a solution. All biochemists know that time spent analysing an impure protein is time wasted. Starting from pure proteins, amino acid sequences and evolutionary relationships between proteins in diverse organisms allow investigation of a protein's biochemical function.

By subjecting protein mixtures to a series of separations, each based on a different property, a pure protein is yielded. Affinity chromatography, of the several sophisticated processes now available to biochemists, takes advantage of the high affinity of many proteins for specific chemical groups.

Affinity chromatography-each of the techniques listed separates proteins from each other and from other biologic molecules based upon characteristics such as size, solubility, and charge. however, only affinity chromatography can use the high affinity of proteins for specific chemical groups or the specificity of immobilized antibodies for unique proteins. in affinity chromatography, a specific compound that binds to the desired proteinsuch as an antibody, a polypeptide receptor, or a substrateis covalently bound to the column material. a mixture of proteins is added to the column under conditions ideal for binding the protein desired, and the column is then washed with buffer to remove unbound proteins. the protein is eluted either by adding a high concentration of the original binding material or by making the conditions unfavorable for binding (e.g., changing the ph). the other techniques are less specific than affinity binding for isolating proteins. dialysis separates large proteins from small molecules. ion exchange chromatography separates proteins with an overall charge of one sort from proteins with an opposite charge (e.g., negative from positive). gel filtration chromatography separates on the basis of size. electrophoresis separates proteins on the principle that net charge influences the rate of migration in an electric field.