نبذه عن الكتاب: 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 raise a polyclonal antibody. At the extreme end of the scale, if the objective is to obtain limited sequence information from the N-terminus of a protein as a preliminary to the design of an oligonucleotide probe for clone screening, then using modern microsequencing techniques, a few micrograms will be sufficient. In the field of proteomics, previously analytical techniques have become preparative with mass spectrometry commonplace for sensitive protein characterization from spots on gels. Chapters 36 and 40–42 describe these methodologies. These different requirements for quantity may well dictate the source of the protein chosen (see Subheading 1.4.) and will certainly influence the approach to purification. Purification of large quantities of protein requires use of techniques, at least in the early stages, that have a high capacity but low resolving power, such as fractional precipitation with salt or organic solvents (see Chapter 13). Process only when the volume and protein content of the extract has been reduced to manageable levels, methods of medium resolution and capacity, such as ion-exchange chromatography (see Chapter 14) can be used leading on, if necessary, to high-resolution