Biomethods – serie

2.1 Introduction ...32 2.2 Technical Procedures ...32 Chemical Synthesis ...35 2.3 Results and Discussion...38 ...Chemical Synthesis ...38 Binding to anti-CCK antisera and CCK-B receptors ...40 Binding to streptavidin agarose and photoelution ...40 Affinity chromatography ...41 2.4 Troubleshooting...43 References ...44 ...Purification of the Receptor for Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) using Biotinylated Ligands ...45 Summary ...45 3.1 Introduction ...46 ...3.2 Technical Procedures ...48 Preparation of a Biotinylated Ligand ...48 Preparation of Membrane Fraction ...50 Solubilization of the PACAP Receptor ...51 Partial Purification of the PACAP Receptor ...52 Affinity Purification of the PACAP Receptor ...54 Final Purification of the PACAP Receptor ...56 Receptor-Binding Assay ...57 3.3 Results and Discussion...58 ...3.4 Troubleshooting ...61 Impurity in PACAP27-Cys-NH...61 2 Low specific activity of the PACAP receptor despite a single band ...62 Acknowledgments ...62 References ...63 ...Photoreactive Biotinylated Peptide Ligands for Affinity Labeling ..." 65 Summary ...65 4.1 Introduction...66 . 4.2 Technical Procedures ..."67 Synthesis of a trifunctional photoactivatable biotinylating reagent ...67 Synthesis of photo reactive biotinylated peptide hormones ...67 Site-specific incorporation of biotin and photo labels in separate steps ...69 Photoaffinity labeling ...72 4.3 Results and Discussion ...76 Synthesis of photoactivatable insulins with permanent biotin labels ...76 Applications: Insulin ...78 VI Examples for other applications ...78 4.4 Troubleshooting...79 References ...81 ...

18. 2 Principle of FACE/Gel Retardation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 18. 3 Labelling of Oligosaccharides with ANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 18. 4 Screening of Carbohydrate Ligands for Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 18. 5 Measurement of Binding Constant for the Interaction Between Protein and ANTS-Labelled Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 18. 6 Measurement of Binding Constant for the Interaction Between Protein and Native Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 ~ The Application of Capillary Affinity Electrophoresis to the Analysis _ of Carbohydrate-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 2 Principle of CAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 19. 3 Determination of Association Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 19. 4 Technical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 19. 5 Limitations of the Technique . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 370 19. 6 Application of CAE to the Analysis of Carbohydrate-Protein Interactions . . . . . . 371 19. 7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 20. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 20. 2 Technical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 20. 3 Sample Detection and Sample Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Autoradiography and staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Sample detection by blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Semipreparative ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 20. 4 Analysis of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Measuring sample mobilities - calculating a retardation coefficient . . . . . . . . . . . . 391 Graphical analysis of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Interpreting ACE patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Reverse ACE . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 20. 5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 XII List of Contributors Nebojsa Avdalovic John T. Gallagher Dionex Corporation Cancer Research Campaign Department of Medical Oncology 445 Lakeside Drive University of Manchester Sunnyvale, CA 94086 Christie CRC Research Centre Klaus Biemann Wilmslow Road Department of Chemistry Manchester M20 4BX Massachusetts Institute of Technology UK Cambridge, MA 02139-4307 USA Geoffrey R.

Twenty five years ago, Bill Stebbins presented the principles of animal psychophysics in an edited volume (Stebbins, 1970) describing an array of modem, creative methodologies for investigating the range of sensory systems in a variety of vertebrate species. These principles included precise stimulus control, a well defined behavioral response, and a rigorous behavioral procedure appropriate to the organism under study. As a generation of comparative sensory scientists applied these principles, our knowledge of sensory and perceptual function in a wide range of animal species has grown dramatically, especially in the field of hearing. Comparative psychoacoustics, i. e. , the study of the hearing capabilities in animals using behavioral methods, is an area of animal psychophysics that has seen remarkable advances in methodology over the past 25 years. Acoustic stimuli are now routinely generated using digital methods providing the researcher with unprecedented possibilities for stimulus control and experimental design. The strategies and paradigms for data collection and analysis are becoming more refined as well, again due in large part to the widespread use of computers. In this volume, the reader will find a modem array of strategies designed to measure detection and discrimination of both simple and complex acoustic stimuli as well experimental designs to assess how organisms perceive, identify and classify acoustic stimuli. Refinements in modem methodologies now make it possible to compare diverse species tested under similar, if not identical, experimental conditions.

Biological processes in any living organism are based on selective interactions be­ tween particular biomolecules. In most cases, these interactions involve and are driven by proteins, which are the main conductors of any life process within the organism. The physical nature of these interactions is still not well known. This book presents an entirely new approach to analysis of biomolecular in­ teractions, in particular protein-protein and protein-DNA interactions, based on the assumption that these interactions are electromagnetic in nature. This new ap­ proach is the basis of the Resonant Recognition Model (RRM), which was devel­ oped over the last 15 years. Certain periodicities within the distribution of energies of delocalised electrons along a protein molecule are crucial to the protein's biological function, i.e. inter­ action with its target. If protein conductivity were introduced, then charges mov­ ing through the protein backbone might produce electromagnetic irradiation or ab­ sorption with spectral characteristics corresponding to energy distribution along the protein. The RRM is capable of calculating these spectral characteristics, which we hypothesized would be in the range of the infrared and visible light. These characteristics were confirmed with frequency characteristics obtained ex­ perimentally for certain light-induced biological processes.

"One should rather go horne and mesh a net than jump into the pond and dive far fishes" (Chinese proverb) Recognizing the precise analytical question and planning the analysis according­ ly is certainly the first prerequisite for successful trace and ultratrace determina­ tions. The second prerequisite is to select the method appropriate to the analyti­ cal specification. The method itself consists of a set of available tools. The third prerequisite is that analysts and operators know the methods weH enough to enjoy challenging themselves as weH as the methods and are rewarded by the joy of high-quality data, fast and economical results and the conviction of having the analytical job under control. This skill is known among analysts or operators working with an exciting new and sometimes complicated analytical technique but is gradually lost on ce a technique becomes "mature" and a routine tool. Unfortunately, laboratory managers often do not allow sufficient training time for their analysts and technicians for "routine" techniques and thus miss an opportunity for motivating their co-workers and obtaining the full benefit of the equipment. Graphite furnace atomic absorption spectrometry (AAS) is one of the mature analytical techniques wh ich is seen as a routine method in most laboratories. More than 10,000 furnaces are operated in elemental trace and ultratrace analy­ ses in laboratories around the world today.

Biomolecular studies are the trial of Man to understand how Nature manages information at the molecular level. The understanding of molecular informa­ tion handling in nature is essential for the molecular optimization in chem­ istry, molecular biology, molecular pharmacology and therefore - as an ex­ ample - for the development of specifically acting drugs. The famous recent method of technical information management is digital electronics. Over the past few years, evidence has arisen that computerized and molecular information managements have many similar and overlapping aspects. For example, both technology and nature use digitized information and both use small structures for the efficient handling of information. Furthermore, they optimize their processes in order to gain a maximum of information with a minimum of invested energy. During the last two decades, novel experimental techniques in biomolec­ ular sciences have paved the way for artificial biomolecular optimization. In the same time interval, the progress of micro system technology has been extended from the field of digital electronics and sensing to micro liquid hand­ ling, and the field of chip-supported substance handling began. It appears that the "marriage" of physical micro technology and molecular processing will be consummated soon. The contact of both fields has been realized in for ex­ ample DNA chips. Such connections will also become relevant in additional fields in the near future. Biomolecular investigations are the first to profit from these fast growing scientific and technical connections between micro systems and molecular sciences.

A Safety Considerations Genomic sequencing involves a number of hazardous steps, such as high current, high voltage, radioactive and highly toxic chemicals. It is, therefore, absolutelyessen- tial that the instructions of equipment manufacturers be followed and that particular attention is paid to the local and federal safety regulations. INTRODUCTION 9 B Introduction During the cloning of genomic DNA many of its characteristics are perma- nently lost. It was therefore necessary to develop a new technique that would give us a closer look at a gene in its normal environment. The powerful technique of genomic sequencing, first described by Church and Gilbert (1984) now makes it possible to have a precise view of a given DNA sequence in a chromosome. This method combines the chemical DNA-sequencing procedure of Maxam and Gilbert (1980) with the detection of DNA sequences by electroblotting and indirect end-labeling by hybridization. Besides studies on the methylation state of single bases in a given gene (Nick et al. , 1986; Saluz and Jost, 1986; Saluz et al. , 1986), genomic sequencing can also be used to study specific DNA-protein interactions in vivo (Church et al., 1985; Giniger et al. , 1985; Becker et al. , 1986; Ephrussi et al. , 1985; Martin et al. , 1986; Nick et al. , 1986; Zinn and Maniatis, 1986).

A Safety Considerations Genomie sequencing involves a number oj hazard- ous steps, such as high eurrent, high voltage, radioaetive and highly toxie chemieals. It is, the- jore, absolutely essential that the instruetions oj equipment manu/aeturers bejollowed and that par- tieular attention is paid to the loeal and jederal safety regulations. I Introduction 13 B Introduction Hypomethylation ofDNA has been positively correlated with thc activation of many eucaryotic genes. During the transition from inactive to active genes changes in the protein/DNA interaction pattern occur. Tran- scriptional activation of eucaryotic genes is mediated by specific interac- tions oftransacting factors with their respective DNA binding sites in Lhe control regions (promoters, enhancers) ofthe genes. This process is ofLen accompanied by changes in local chromatin strucLure, witnessed by the appearance of nuclease hypersensitive sites, as weil as by changes in protein-DNA interactions and, in the case of higher eucaryotes, alterations ofthe cytosine methylation pattern.The sole available experimental tech- nique that permits the study ofthe latter phenomena at single nucleotide resolution is direct genomic sequencing/footprinting, pioneered by Church and Gilbert (1984). This method combines the chemical DNA- sequencing procedure of Maxam amI Gilbert (1980) with thc detection 01' DNA sequences by electroblotting and indirect end-Iabeling by hybridiza- tl0n. An alternative possibility is the novel procedure (Saluz and . lost, 1989), using Taq polymerase. The first steps 01' both meLhods are essen- tially the same: total genomic DNA is digested wiLh a suilable restriction enzyme and the resulting DNA fragments are chemically sequeneed.

This laboratory guide comes at a time when several other method books have already been published in this field. Is this one different from the others? Yes and no. There was no attempt made to be comprehensive. Rather, data were brought to bear on areas where enough competence has been gathered in our laboratories and to complement recent method books (many of which cover extensively various aspects of molecular biology) in those matters which appeared to us somewhat neglected. There was a constant preoccupation and effort to provide miniaturized proce­ dures that are both simple and time-saving. Interest was devoted to standardized procedures and culture conditions, avoiding dogmas such as those giving excessive importance to sophisticated culture media with endless adjustments for local or personal considerations. The key to success is the quality of the plant material serving as a source of cells. Consequently, isolation. extraction or culture techniques can be simplified and standardized. This is symptomatic for our times as it marks the end of a period when methodological matters were frequently above the biological problems. The times of "methods above all" is basically over, despite the fact that many of us still believe that, say, tissue culture is a "science" per se. By presenting a few original techniques we believe that one seriously reduces the empiricism still prevailing in this area of research.

A Safety Considerations Many techniques described here involve a number of hazards, such as high electrical current and voltage, radioactivity and highly toxic chemicals. It is absolutely essential that the instructions of equipment manufacturers be followed, and that particular attention be paid to the local and federal safety regulations. B Introduction The expression of prokaryotic and eukaryotic genes has been shown most often to be regulated at the level of mRNA synthesis. Thanks to the rapid development of methods for dissecting DNA sequences, cis-acting regulatory elements such as promoters and enhancers have been recognised. More recently, the widely expressed intuition that discrete sequences within these elements constitute binding sites for sequence-specific binding proteins has been confirmed, especially through the use of "footprinting" assays (for examples, Galas and Schmitz, 1978). This and similar assays have already resulted in the recognition, isolation and analysis of DNA-bind­ ing proteins for several genes. Excellent reviews exist of the structural studies on these transcription regulatory proteins and related DNA elements (for example, Glover, 1989 and Johnson and McKnight, 1989), to which the reader is referred for detailed information. To set the scene for applications of the techniques described in this volume, only the barest outline of previous studies is presented here. Protein-DNA interactions are dependent on very specific tertiary configurations of the binding protein which allow the closest contact with the DNA helix.