bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic analysis of the interaction between an analyte in solution and an immobilized protein
Authors: Karlsson R , Löfås S 
Published: November 2023

Abstract
Proteins function by interacting with other biomolecules. On a molecular basis the interaction can be characterized in terms of the affinity and the kinetics of the interaction. The affinity is often easy to measure (1) and describes the strength of the interaction. Kinetic analysis is more informative than affinity analysis and gives additional data regarding the rate of complex formation and dissociation. When kinetic analysis is possible, the binding assay is facilitated and fewer experiments can be performed. This is illustrated in Figure 1. Here data obtained using a traditional solid phase assay (i.e. an ELISA) and a biosensor-based kinetic assay is compared. When a single concentration of analyte is used a traditional assay gives one data point that in itself is almost meaningless. In contrast a biosensor assay can provide direct and continuous information on how the analyte binds to and dissociates from the immobilized ligand. In theory a single binding curve can be used to determine both the association rate constant, ka, and the dissociation rate constant, kd.. The affinity constant KA can then be calculated from the ratio between ka and kd.
Peptide mapping
Authors: Andrews Anthony T 
Published: November 2023

Abstract
Current versions of one-dimensional techniques such as polyacrylamide gel electrophoresis (PAGE), polyacrylamide gel electrophoresis in the presence of detergents such as sodium dodecyl sulfate (SDS-PAGE), immuno electrophoresis (IE), and isoelectric focusing in polyacrylamide gels (IEF) have very high resolution, are reproducible, reliable, rapid, and relatively inexpensive and simple to set-up and use. Although slower and more complex two-dimensional separations give unparalleled resolution that no other analytical method can match, the number of samples which can be examined in a given time is much reduced. In spite of these capabilities, however, there are situations where neither one-nor two-dimenstional methods can give an unequivocal answer. Most of these devolve into a question of ‘relatedness’ between two or more proteins and this is where peptide mapping can make a major contribution. When working with intact proteins, identifying an un known or following changes in protein components typically involves comparison with known standard proteins.
Physical Methods Of Study: Differential Scanning Calorimetry
Authors: Taylor Kevin M G , Craig Duncan Q M 
Published: November 2023

Abstract
Thermal methods, and in particular differential scanning calorimetry (DSC), are well established within the liposome field, and are most frequently used to measure the phase behaviour of the phospholipid bilayers, from which information on phospholipid conformation, bilayer fluidity, drug-liposome interactions, and rate of drug release may be derived. In addition, knowledge of the transition behaviour of phospholipids is essential for the rational development of manufacturing protocols. There is also a growing interest in the use of thermal methods for the examination of freeze-dried liposomes, particularly in terms of characterizing the glass transitional behaviour of these systems.
Fluorescence Methods in Liposome Research
Authors: Düzgünes Nejat , Bagatolli Luis A , Meers Paul , Oh Yu-Kyuong , Straubinger Robert M 
Published: November 2023

Abstract
Liposomes of certain compositions can undergo fusion in the presence of the appropriate ionic conditions and pH, or fusion-inducing proteins or peptides (1-4). Thus the molecular requirements for membrane fusion can be explored systematically, using assays that monitor either the intermixing of membrane components or the coalescence of internal aqueous contents of liposomes. In this section we describe widely used fluorescence assays that monitor the kinetics of the fusion process.
Single-strand conformation polymorphism analysis
Authors: Hayashi Kenshi , Kukita Youji , Inazuka, Masakazu , Tahira Tomoko 
Published: November 2023

Abstract
SSCP (single-strand conformation polymorphism) is widely used for mutation detection because of its simplicity and versatility. In this method, a segment to be searched for a mutation is amplified by PCR from genomic DNA or cDNA, denatured, and separated by electrophoresis in media having sieving properties without denaturant (1). Mutation usually causes a mobility shift in electrophoresis, and so, is detected. Conformational change of the single stranded mutant DNA is believed to be the reason for the mobility shift.
The theory and practice of in situ hybridization
Authors: Wilkinson David G 
Published: November 2023

Abstract
The in situ detection of nucleic acid sequences, whether of genes on chromosomes or viruses, or of mRNA in tissues, provides a direct visualization of the spatial location of specific sequences that is crucial for elucidation of the organization and function of genes. As a consequence, methods of in situ hybridization have become important techniques in a number of fields, including diagnosis of chromosomal rearrangements, detection of viral infections, and analysis of gene function during embryonic development. In situ hybridization takes advantage of the specific annealing of complementary nucleic acid molecules, which can be DNA and/or RNA, through hydrogen bonds formed between bases attached to the sugar-phosphate backbone: adenine (A) anneals with thymine (T, in DNA) or uracil (U, in RNA), and cytosine (C) anneals with guanine (G). This base pairing underlies the formation of a double-stranded complex, in which one· strand has the opposite orientation to the other with respect to the sugar-phosphate backbone (Figure JA); the sequence is read from 5’ to 3’ (this refers to the positions of the sugar at which the phosphate residues are attached). Any nucleic acid sequence can therefore be specifically detected by use of a probe that is the ‘antisense’ reverse complementary sequence. An important practical asset is the speed with which such probes can be generated by the cloning of DNA sequences, amplification by the polymerase chain reaction (PCR), or the synthesis of oligonucleotides.
Oligonucleotide probes for in situhybridization
Authors: Rattray Marcus , Michael Gregory J 
Published: November 2023

Abstract
Oligonucleotide probes are convenient for determining the sites of gene expression using in situ hybridization. Since the probes are synthetic, their use does not require much specialized equipment for molecular biology, the growth of bacteria, or the purification of nucleic acids. Oligonucleotide probes can be designed to form complementary base pairs with a target sequence, that can be RNA or DNA. In this chapter we consider binding of oligo¬ nucleotide probes to RNA targets only. The probe is labelled so that its presence on tissue sections can be detected. Probe is applied to the tissue under conditions that hybridization takes place, and then the sections are washed to remove non-specific binding. After these steps have taken place, the presence of the probe can be detected by autoradiography (for radio¬ labelled probes) or by histochemistry (for non-radiolabelled probes). It is possible to use oligonucleotide probes to measure the level of gene expression in individual cells or in tissues using appropriate image analysis techniques.
Electron microscope in situ hybridization: the non-isotopic post-embedding procedure
Authors: Puvion-Dutilleul Francine 
Published: November 2023

Abstract
Although the advent of in situ hybridization for light microscopy goes back almost three decades (1, 2) and its extention to the electron microscope (EM) level occurred shortly after, it is only recently that this technique revolution¬ ized our knowledge about the spatial distribution of defined nucleic acid molecules in the cell. Binder et al. (3) broke new ground in this field by con¬ comitantly using for the first time hydrosoluble resin-embedded material, non-isotopic probes, and immunogold labelling of hybrids. This post embedding non-radioactive procedure was immediately adopted, and became widely used after some procedural improvements which rendered it a method applicable to a wide variety of biological systems.
Microscopy of chromosomes
Authors: Sumner A T , Leitch A R 
Published: November 2023

Abstract
Chromosomes have been studied for something like 120 years, and for most of this period they were visualized using uniform staining with a variety of dyes. This was adequate for studies of the gross structure and behaviour of chromosomes, but identification of individual chromosomes was difficult, being based only on chromosome morphology and size, and specific chromosome substructures could not be studied. Just over 25 years ago, two new techniques emerged that revolutionized the study of chromosomes: (a) Chromosome banding, a series of staining techniques that produced patterns of longitudinal differentiation on chromosomes, and allowed identification of specific chromosomes and chromosomal substructures. (b) In situ hybridization, which labels specific DNA sequences in chromosomes.
Nanovid microscopy
Authors: Lee Greta M 
Published: November 2023

Abstract
Nanovid microscopy, nanometre-sized particles viewed with video-enhanced microscopy (1-3) is used to directly observe the lateral mobility of gold labelled membrane proteins and lipids, to observe the precise location and mobility of gold labelled pericellular and extracellular matrix macromolecules, and to follow endocytosis of gold labelled molecules. The technique involves the use of 30--40 nm colloidal gold particles conjugated to antibodies, streptavidin, lectins, or other ligands. The association of the antibody to the gold is non-covalent but extremely stable. Since a single colloidal gold particle is typically conjugated to many antibody molecules (4), an individual gold particle may bind to a small group of like membrane molecules rather than to a single molecule (5). The gold tag is visualized using video contrast enhancement to detect the light scattering of the gold particle. The gold can be viewed with bright-field, DIC, or epipolarization optics. Examples of bright-field and DIC images are shown in Figure 1.
Embryonic feeder cells
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
To maintain their pluripotency, ES cells are usually cultured either on plastic plates (optionally can be gelatinized) in the presence of leukemia¬ inhibitory factor (LIF) or on a layer of mitotically inactive feeder cells derived from embryonic fibroblasts. These embryonic feeder, or EF, cells are prepared from day 13-14 embryos., We culture our ES cells on mitomycin C-treated primary EF cells and in the presence of LIF.
Preparation of EF cells obviously needs to be done in advance of any ES cell work. EF cells must be G418-resistant (or resistant to any other selection drug you wish to use in isolating homologous recombinants) and, therefore, must be prepared from mice harboring the neo gene. We use a 129-derived strain which harbors pSV2neo (135).
Chapter 16
Authors: picking ES clone 
Published: November 2023

Abstract
The number of clones that must be recovered will depend on a number of factors such as the type of selection (e.g. selection for homologous recombinants and against random integrants), number of colonies obtained after selection, method of screening for homologous recombinants (Southern vs PCR), knowledge about the targeting frequency for the locus of interest, etc. For obvious reasons, however, the more clones isolated the higher the probability of recovering a homologous recombinant. In situations in which we have targeted the same locus in multiple instances (e.g. the immunoglobulin heavy chain locus) we have generally observed a similar frequency of homologous recombination events and therefore know approximately how many colonies must be isolated to obtain a homologous recombinant. As a very general number, at least 250-300 colonies should be isolated for examination of a homologous recombination event.
Embryo aggregation
Authors: Torres Raul M , Kühn Ralf 
Published: November 2023

Abstract
For embryo (morula) aggregation less equipment is needed than for blastocyst injection: required are a stereomicroscope (∼40x magnification) with transmission light for setting up aggregates and a CO2 incubator for culture so that researchers setting up a new lab may choose aggregation instead of blastocyst injection for the production of chimeric mice. For embryo aggregation eight-cell stage embryos up- to morulas should be isolated from the oviduct of superovulated or normally mated females at day 2.5 p.c. After removal of the zona pellucida the embryos are placed individually, together with a small clump of about 10 − 20 ES cells, into small ‘aggregation wells’ of a plastic dish for aggregation (aggregation plate). After overnight culture the majority of these aggregates develop into compacted morulas or early blastocysts which are then transferred into the uteri of pseudopregnant females in the same way as microinjected blastocysts (see Chapter 27). Our protocol is adapted from the one described by Nagy and Rossant (170) except that only one embryo is used per aggregate and that media contain a low concentration of phenol red.
Arf
Authors: Roth Michael G 
Published: November 2023

Abstract
The six mammalian ADP-ribosylation factors form a distinct family within the Ras GTPase superfamily (1). The amino-acid sequences of these 20 kDa guanine nucleotide-binding proteins are at least 70% identical in plants, protozoa, and animals, attesting to their importance in all eukaryotic cells. Two genes encode redundant Arf proteins in S. cerevisiae that are essential for vegetative growth and secretory function (2). A third gene, ARF3, encoding a protein 52% identical to Arf1p, cannot compensate for the loss of ARF1 and ARF2 (1). In mammalian cells, the major function of Arfs is to regulate a variety of membrane transport processes, although Arf proteins also activate phospholipase D enzymes (3, 4) and one member of the family, Arf6, may be involved in regulating the actin cytoskeleton (5). Sarl, a small GTPase that is 37% identical to Arf1, functions in the assembly of the COPII coated vesicles that are responsible for membrane transport from the endoplasmic reticulum to the Golgi complex (6). The molecular details of Sar1 function are better understood than those of Arf proteins (7) and Sar1 has therefore been used as a model for understanding Arfs, particularly Arf1. However, this is likely to be misleading, since the regulation of Arfl is clearly quite different from that of Sarl, and Arfl appears to have a greater number of activities than Sarl.
Proline isomerization and its catalysis in protein folding
Authors: Balbach Jochen , Schmid Franz X 
Published: November 2023

Abstract
In proteins the peptide bonds are usually in the trans conformation, which is favoured energetically over cis in both folded and unfolded proteins. However, an appreciable proportion of the peptide bonds preceding proline (prolyl bonds),* and occasionally those preceding other amino acids, arc in the strained cis conformation. These cis peptide bonds create a problem for protein folding. The non-native trans forms predominate at equilibrium in the unfolded or nascent protein molecule and, since cis⇌trans isomerization is an intrinsically slow reaction — because rotation about a partial double bond is required — the rate of folding to the native state is governed by the rate of isomerization. Non-native prolyl isomers do not block the early stages of folding of a protein chain, but they markedly retard the overall folding process. This is clearly seen in the case of small, single-domain proteins. Many of them refold within a few milliseconds when they contain native prolyl isomers, but when non-native isomers are present (in particular trans isomers of bonds that are cis in the native state) folding is retarded from the millisecond to the minute time range.
Measurement of cytoplasmic and nuclear antigens
Authors: Larsen J0rgen K 
Published: November 2023

Abstract
1 Introduction
Flow cytometry has become the technique of choice for the quantification of a variety of intracellular molecules involved in the regulatory pathways of cellular proliferation, differentiation, and death, as well as in signal transduction and cytokine production. This development is based on recent improvements of the immunochemical methods for detecting target antigens within the cytoplasm and nucleus, supported by the increased assortment of commercially available specific antibodies and fluorochrome conjugates, new signal amplification methods, and improved instrumentation of multiparameter data acquisition and analysis.
Analysis of DNA-measurement of cell kinetics by the bromodeoxyuridine/ anti bromodeoxyuridine method
Authors: Wilson George D 
Published: November 2023

Abstract
Cell kinetics is primarily concerned with the measurement of time parameters in the growth of biological systems. This definition sets it apart from other techniques involved in the study of cellular proliferation. Most methods involve a ‘snapshot’ assessment of cells in a particular phase of the cell cycle (e.g. mitotic index, tritiated thymidine labelling index or S phase fraction from DNA histograms) or cells actively involved in a proliferation cycle (e.g. Ki-67, PCNA staining-see Chapter 9). Whilst static parameters of cell proliferation can be informative, they give no indication of the rates of cell-cycle progression and are subject to ambiguity as will be discussed later in this chapter.
Nucleic acid isolation and analysis
Authors: Talbot Nicholas J 
Published: November 2023

Abstract
The ability to carry out molecular genetic analysis of filamentous fungi is very heavily dependent on being able to extract and purify large amounts of DNA and RNA from fungal cells. Many procedures have been devised in order to purify nucleic acids from eukaryotic cells and a large number of commercially available kits have been formulated for this purpose. Most procedures devised, however, have been optimized for use in cells of model organisms such as yeast, Arabidopsis, Drosophila, and human cells. Adapting these procedures for use in filamentous fungi produces highly variable results and some species have proved recalcitrant to molecular analysis largely due to workers being unable to purify high-quality nucleic acid samples.
DNA-mediated transformation of fungi
Authors: Bowyer Paul 
Published: November 2023

Abstract
The advent of techniques for manipulating and isolating genes from plant pathogenic fungi has lead to a rapid increase in our understanding of how these organisms cause disease (1). An important factor in these advances has been the ability to re-introduce homologous or heterologous genes into these organisms. Transformation systems for model fungi such as yeast, Neurospora crassa, and Aspergillus nidulans, have existed since the 1970s and these techniques are being adapted for use in a growing array of pathogens. This chapter concentrates on techniques developed for pathogenic fungal species, but the techniques have often been found to be broadly applicable among fungal species. The vast majority of transformation protocols for filamentous fungi are based on permeabilizing cell membranes with polyethylene glycol (PEG), or electroporation.
Investigations of Oomycete cell biology
Authors: Hardham Adrienne R 
Published: November 2023

Abstract
The Oomycetes are a distinct group of organisms which, like many true fungi (Basidiomycetes, Ascomycetes, and Zygomycetes), form filamentous hyphae during vegetative (somatic) growth. They are also heterotrophs with absorptive nutrition. Many characteristics of Oomycete organisms, however, indicate that they are not true fungi but are most closely related phylogenetically to the Chromophyte algae and other flagellate protists which possess tripartite flagellar hairs (1–3).
The cytogenetics of pregnancy
Authors: Boyle T , Griffin D 
Published: November 2023

Abstract
Prenatal cytogenetic diagnosis is made possible by the in vitro culture of human cells and tissues. Amniocentesis is the removal of amniotic fluid usually performed between 15 and 17 weeks’ gestation, although sampling has been carried out as early as 12 weeks (1). Chorionic villus sampling allows for first trimester analysis with transcervical or transabdominal aspiration of villi performed at 10-11 weeks’ gestation. Solid tissue specimens may be examined at various stages of gestation and encompass both fetal and extra-embryonic tissues. The techniques supplied are also applicable to adult skin biopsy.
At the time of writing, the cytogenetic study of pre-implantation embryos is a promising new field and, together with advances in the study of sperm and oocyte chromosomes, may provide further help to couples with infertility problems or pregnancies at high risk of genetic disease.
Microscopy and image analysis systems
Authors: Levy E R , Maher E J 
Published: November 2023

Abstract
The microscope is one of the most important tools for a cytogeneticist. It is essential that the microscope provides contrast and resolution of the sample, along with suitable magnification, and to achieve these, three criteria must be met:
the lens system must reveal the required detail, i.e. provide adequate resolution
the image must have sufficient contrast to allow the eye to distinguish what has been resolved
the magnification must be sufficient so that the image of separate points resolved by the microscope fall upon separate cones of the retina.
The aim of this chapter is to provide enough information for the beginner to be able to understand the workings of both light and fluorescence compound microscopes, and to be able to set up and maintain them.
Micromanipulation by laser microbeam and optical tweezers
Authors: Greulich Karl Otto 
Published: November 2023

Abstract
In several chapters at the beginning of this book, techniques were described using light for observation of, and making measurements on cells. This chapter will describe techniques, using light not only for observation but also for manipulation. Here the fact that light is a carrier of energy (heat) and a carrier of momentum (force) is exploited. With the heat effect one can perforate plant cells, cut subcellular structures such as cytoplasmic strands or chromosomes, and can fuse plant protoplasts in a controlled manner. With the force effect it is possible to hold or move cells and subcellular structures as if light were a pair of tweezers, which in contrast to conventional tweezers can also work in the interior of closed objects. Books on laser microbeams and optical tweezers (1) or solely on optical tweezers (2) and a number of reviews (for example refs 3-7) are available in the literature.
Electron microscopy
Authors: Hawes Chris , Satiat-Jeunemaitre Béatrice 
Published: November 2023

Abstract
Despite the recent resurgence in the use of light microscopy (LM) due to the popularity of the confocal microscope coupled with the wide availability of image processing techniques (see Chapters 1 and 2), the unparalleled resolution offered by the electron microscope will maintain its importance as a basic tool in cell biology. Recent developments in preparative protocols, such as ultra-rapid freeze-fixation and freeze-substitution have permitted the preservation of cellular ultrastructure with great fidelity. Whilst the use of immunogold label ling techniques permit the location of antigens within cells at a resolution not possible by LM or confocal based immunocytochemical techniques.