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A comprehensive collection of essential, time-tested recipes for successful protein fractionation and purification in any experimental circumstance. The protocols give step-by-step instructions on how to select a source for the protein of interest, how to obtain a usable initial extract, how to purify the protein from that extract using both chemical and molecular methods, and how to dry and store the purified protein. Protein Purification Protocols provides all that is needed to design and carry out a successful purification program. It helps both experienced and novice investigators to clarify and define their purification problems and then provides a comprehensive set of tools for a practical solution.
E-Book Content
CHAPTER General 1 Strategies Shawn Doonan 1. Defining the Problem The chapters that follow in this volume give detailed instructrons on how to use the various methods that are available for purification of proteins. The question arises, however, of which of these methods to use and in which order to use them to achieve purification in any particular case. That is, the purification problem must be clearly defined. What follows outhnes the sorts of questions that need to be asked as part of that definition and how the answers affect the approach that might be taken to developing a purification schedule. It should be noted here that the discussion does not touch on the special cases of purification of proteins at industrial scale or for therapeutic applications; these raise very specific problems that are outside the scope of this chapter (see refs. I and 2, respectively, for a coverage of these topics). 1.1. How Much Do I Need? The answer to this question depends on the purpose for which the protein is required. For example, to carry out a full chemical and physical analysis of a protein may require several hundreds of milli