Which of the following techniques for purification of proteins can be made specific for a given protein?
Ans. B. Affinity chromatographya. Each of the techniques listed separates proteins from each other and from other biologic molecules based on 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.b. In affinity chromatography, a specific compound that binds to the desired protein--such as an antibody, a polypeptide receptor, or a substrate--is covalently bound to the column material.c. 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.d. 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).e. 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.f. Electrophoresis separates proteins on the principle that net charge influences the rate of migration in an electric field.
Dialysis
Affinity chromatography
Gel filtration chromatography
Ion exchange chromatography
Structure & Function of Protein