TABLE OF CONTENTS This unit introduces the student to the basic steps in a protein expression, extraction, and purification scheme. Developing a protein purification scheme involves optimizing the methods to best maintain and improve the functional activity of the macromolecule. This requires minimizing the number of consecutive methods employed, possibly varying their order, and testing different elution conditions and reagents. It is common to systematically vary the gradient conditions by adjusting lower and upper salt concentrations. Specification concentrations (e.g., Mg 2+) and pH are also manipulated. Teaching students how to approach the purification of a previously uninvestigated protein system involves describing each of the different types of substeps and then explaining how each variable can be manipulated to obtain pure protein. Revealing "hidden" inhibitors and/or problems caused by interactions between two or more components can also lead to success. Another important goal is sample preservation. Reviewing the points made in the Introductory Unit will refresh some important caveats to keep in mind when trying to optimize the procedures. These ideas are addressed in more detail in appendices in this Unit that discuss protein manipulations in preparation for crystallographic and protein folding studies.
One can present a reasonably useful model for a general approach to characterize the purity of a material and some important quantitative aspects to consider when doing so. Students should realize that this manual merely introduces the subject. The broad range of assays used to characterize the diverse activities of proteins is...
