This open-end treatise on methods concerning pro tein separation had its beginning in an American Chemical Society symposium entitled "Con temporary Protein Separation Methods" which was held in Atlantic City, New Jersey in September 1974. The purpose of the symposium-and subse quently of the present work-was to review the available modern techniques and underlying principles foer achieving one of the very important tasks of experimental biology, namely the separation and characterization of proteins present in complex biological mixtures. Physicochemical characterization was covered only as related to the parent method of fractionation and there fore involved mostly mass transport processes. Additionally, the presentation of methods for gaining insight into complex interacting protein profiles was considered of paramount importance in the interpretation of separation patterns. Finally, specific categories of proteins (e. g. , chemically modified, deriving from a specific tissue, conjugated to different moieties, etc. ) require meticulous trial and selection and/or modification of existing methodology to carry out the desired separation. In such cases, the gained experience provides valuable guidelines for further experimentation. Although powerful techniques exist today for the separation and related physicochemical characterization of pro teins, many biological fractionation problems require further innovations. It is hoped that the description in the present treatise of some of the available separation tools and their limitations will provide the necessary integrated background for new developments in this area. Nicholas Catsimpoolas Cambridge, Massachusetts vu CONTENTS Contents of Volume 1 . xvii Chapter 1 Scanning Gel Cbromatography Gary K. Ackers I.

It has now been over a decade since isoelectric focusing became estab lished as a valuable addition to our arsenal of physical methods for separa tion of proteins and other amphoteric substances. The high resolving power of the technique and its unique ability to separate as well as concentrate have been amply demonstrated in several international symposia. The past few years have led to the wide acceptance of standardized methodologies for analytical and preparative purposes. These procedures afford rapid, reproducible separations that have led to a dramatic increase in our under standing of many areas of biological and biomedical research. Conse quently, we considered it desirable to review some of the important appli cations of the isoelectric focusing technique. To accomplish this, we have solicited the participation of noted authorities to highlight major progress in their fields of expertise. Thus, the material in this book will emphasize recent advances in knowledge rather than methodological aspects, except when special procedures are reviewed. Nicholas Catsimpoolas James W. Drysdale vii Contents Chapter J Isoelectric Focusing of Human Saliva, Cerebrospinal Fluid, and Urine Josie A. Beeley I. Introduction I II. Isoelectric Focusing of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A. Gel Rods and Thin Layer Gels . . . . . . . . . . . . . . . . . . . . . . . . 2 B. Liquid Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 C. a-Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 D. Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 E. Blood Group Specific Substances . . . . . . . . . . . . . . . . . . . . . 11 F. Vitamin B12 Binding Proteins (Cobalophilins) . . . . . . . . . . . 12 G. Other Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 H. Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 I. Specific Salivary Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 J. Dental Plaque . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 K. Other Proteins . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . 16 III. Isoelectric Focusing of Cerebrospinal Fluid . . . . . . . . . . . . . . . ."

The selective combination of physical, biochemical, and immunological prin ciples, along with new knowledge concerning the biology of cells and advance ments in engineering and computer sciences, has made possible the emergence of highly sophisticated and powerful methods for the analysis of cells and their constituents. This series on Cell Analysis is, therefore, aiming at providing the theoretical and practical background on how these methods work and what kind of information can be obtained. Cell Analysis will cover techniques on cell separation, cell identification and classification, characterization of orga nized cellular components, functional properties of cells, and cell interactions. Applications in cell biology, immunology, genetics, toxicology, specific diseases, diagnostics and therapeutics, and other areas will be covered whenever relevant results exist. Nicholas Catsimpoolas Boston, Massachusetts vii Contents Chapter I Quantification of Red Blood Cell Morphology James W. Bacus I. History .. II. Details of Red Cell Measurements. 3 III. Cell Sample Population Distributions. 11 IV. Discussion and Summary. 25 References. 30 Chapter 2 Laser Microirradiation and Computer Video Optical Microscopy in Cell Analysis Michael W. Berns and Robert J. Walter I. Introduction 33 II. Laser Microbeams 34 III. Computer-Enhanced Video Microscopy for Laser Microsurgery."

Presently, the need for methods involving separation, identification, and characterization of different kinds of cells is amply realized among immu nologists, hematologists, cell biologists, clinical pathologists, and cancer researchers. Unless cells exhibiting different functions and stages of differ entiation are separated from one another, it will be exceedingly difficult to study some of the molecular mechanisms involved in cell recognition, spe cialization, interactions, cytotoxicity, and transformation. Clinical diagno sis of diseased states and use of isolated cells for therapeutic (e. g., immu notherapy) or survival (e. g, transfusion) purposes are some of the pressing areas where immediate practical benefits can be obtained by applying cell separation techniques. However, the development of such useful methods is still in its infancy. A number of good techniques exist based either on the physical or biological properties of the cells, and these have produced some valuable results. Still others are to be discovered. Therefore, the purpose of this open-ended treatise is to acquaint the reader with some of the basic principles, instrumentation, and procedures presently in practice at various laboratories around the world and to present some typical applications of each technique to particular biological problems."