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DNA Repair Protocols
The first edition of this book, published in 1999 and called DNA Repair Protocols: Eukaryotic Systems, brought together laboratory-based methods for studying DNA damage and repair in diverse eukaryotes: namely, two kinds of yeast, a nematode, a fruit fly, a toad, three different plants, and human and murine cells. This second edition of DNA Repair Protocols covers mammalian cells only and hence its new subtitle, Mammalian Systems. There are two reasons for this fresh emphasis, both of them pragmatic: to cater to the interests of what is now a largely mammalocentric DNA repair field, and to expedite editing and prod- tion of this volume. Although DNA Repair Protocols: Mammalian Systems is a s...
Cell Imaging Techniques
A diverse collection of state-of-the-art methods for the microscopic imaging of cells and molecules. The authors cover a wide spectrum of complimentary techniques, including such methods as fluorescence microscopy, electron microscopy, atomic force microscopy, and laser scanning cytometry. Additional readily reproducible protocols on confocal scanning laser microscopy, quantitative computer-assisted image analysis, laser-capture microdissection, microarray image scanning, near-field scanning optical microscopy, and reflection contrast microscopy round out this eclectic collection of cutting-edge imaging techniques now available. The authors also discuss preparative methods for particles and cells by transmission electron microscopy.
Mast Cells
A cutting-edge collection of readily reproducible techniques for the isolation, culture, and study of activation and signaling in human mast cells. These methods take advantage of the latest advances in molecular biology, technology, and information science. They include methods for the identification of mast cells, the development of mast cells in vitro, the study of mast cell signaling and gene expression, and the measurement of mast cell expression of inflammatory mediators. Additional chapters cover methods for studying mast cell interactions with other cell types (endothelial cells, fibroblasts, and B cells), the roles of mast cells in host defense, and mast cell apoptosis.
Nuclear Reprogramming
A wide-ranging collection of readily reproducible methods for performing nuclear reprogramming by nuclear transfer in several different species, by fusion through both chemical treatment and electrically shocking cells, and by in vivo treatment of cells with cell extracts. Several methods of monitoring nuclear reprogramming are also presented, including the use of transgenic markers, activation of telomerase as an ES-specific marker, light and electron microscopic observation of structural changes in the nucleus, and verification of surface marker expression and the differentiation potential of stem cells. Biochemical methods are provided for the examination of chromatin protein modifications, nucleosomal footprinting, transcription factor binding, and the study of DNA methylation changes both at the specific locus level and at the level of the whole nucleus.
Human Embryonic Stem Cell Protocols
A comprehensive collection of diverse techniques for the molecular and cellular manipulation of human embryonic stem (hES) cells. These readily reproducible methods have been optimized for the derivation, characterization, and differentiation of hES cells, with special attention given to regenerative medicine applications. A companion CD provides color versions of all illustrations in the book. The protocols follow the successful Methods in Molecular BiologyTM series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Human Retrovirus Protocols
A cutting-edge collection of basic and state-of-the-art methods optimized for investigating the molecular biology of this class of retrovirus. These readily reproducible techniques range from methods for the isolation and detection of human retroviruses to cutting-edge methods for exploring the interplay between the viruses and the host. Here, the researcher will find up-to-date techniques for the isolation and propagation of HIV, HTLV, and foamy virus from a variety of sources. There are also assays for determining the cell tropism of HIV-1, the coreceptor usage of HIV-1, and human gene expression with HIV-1 infection by microarrays, as well as for phenotyping HIV-1 infected monocytes and examining their fitness. Highlights include the detection and quantification of HIV-1 in resting CD4+, a new cloning system for making recombinent virus, cDNA microarrays, and the determination of genetic polymorphisms in two recently identified HIV-1 co-factors that are critical for HIV-1 infection.
RNA Silencing
A collection of readily reproducible methods for the design, preparation, and use of RNAs for silencing gene expression in cells and organisms. The techniques range widely and include methods addressing the biochemical aspects of the silencing machinery, RNA silencing in non-mammalian organisms, and the in vivo delivery of siRNAs and silencing vectors. There are also techniques for designing, preparing, and using RNAs to silence gene expression, for fine-tuning regulation by targeting specific isoforms of a given gene, and for the study and use of microRNAs. The protocols follow the successful Methods in Molecular BiologyTM series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Plant Cell Culture Protocols
A comprehensive state-of-the-art collection of the most frequently used techniques for plant cell and tissue culture. Readily reproducible and extensively annotated, the methods range from general methodologies, such as culture induction, growth and viability evaluation, and contamination control, to such highly specialized techniques as chloroplast transformation involving the laborious process of protoplast isolation and culture. Most of the protocols are currently used in the research programs of the authors or represent important parts of business projects aimed at the generation of improved plant materials. Two new appendices explain the principles for formulating culture media and the composition of the eight most commonly used media formulations, and list more than 100 very useful internet sites.
Chemical Genomics
Chemical genomics is an exciting new field that aims to transform biolo- cal chemistry into a high-throughput industrialized process, much in the same way that molecular biology has been transformed by genomics. The inter- tion of small organic molecules with biological systems (mostly proteins) underpins drug discovery in the pharmaceutical and biotechnology industries, and therefore a volume of laboratory protocols that covers the key aspects of chemical genomics would be of use to biologists and chemists in these orga- zations. Academic scientists have been exploring the functions of proteins using small molecules as probes for many years and therefore would also b- efit from sharing idea...
In Situ Hybridization Protocols
The technique of in situ hybridization, in its various forms, has been used routinely in many laboratories for a number of years. In the post-genome era, gene arrays and proteomics have allowed us to identify hitherto unknown unrecognized pathways and mechanisms. However, rather than diminish the importance of in situ hybridization, the now widespread use of screening te- nologies has increased the need to temporally and spatially localize the dist- bution of mRNA expression. Our intention, in In Situ Hybridization Protocols is to provide ample inf- mation for novices planning to set up the in situ hybridization technique and use it in their laboratory for the first time, as well as giving u...
