E-Book Overview
This new edition of Protein Purification Protocols (1996) completely updates the existing protocols to reflect recent advances and adds the enormous new array of proteomic techniques for protein isolation and analysis. These cutting-edge techniques include not only two-dimensional gel electrophoresis for analysis and characterization, but also analytical chromatography for multidimensional separations of proteins and peptides, and mass spectrometry for isolating proteins.
E-Book Content
Methods in Molecular Biology TM TM VOLUME 244 Protein Purification Protocols Second Edition Edited by Paul Cutler 1 General Strategies Shawn Doonan and Paul Cutler 1. Defining the Problem The chapters that follow in this volume give detailed instructions 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 outlines the sorts of question 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 concentrates mainly on laboratory-scale isolation of proteins. Special cases of purification of therapeutic proteins and isolation at industrial scale are covered in Chapters 43 and 44 (1–5). 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 milligrams of purified material, whereas a kinetic analysis of the reaction catalyzed by an enzyme could perhaps be done with a few milligrams and less than 1 mg would be required to