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Functional Genomics
This collection of robust, readily reproducible methods for microarray-based studies includes expert guidance in the optimal data analysis and informatics. On the methods side are proven techniques for monitoring subcellular RNA localization en masse, for mapping chromosomes at the resolution of a single gene, and for surveying the steady-state genome-wide distribution of DNA binding proteins in vivo. For those workers dealing with massive data sets, the book discusses the methodological aspects of data analysis and informatics in the design of microarray experiments, the choice of test statistic, and the assessment of observational significance, data reduction, and clustering.
Superantigen Protocols
Leading researchers in the biological, chemical, and physical investigation of superantigens describe in step-by-step detail their best experimental techniques to assess the physical characteristics and biological effects of superantigens. Their protocols range from those for investigating the interactions of superantigens with cellular receptors to those for the analysis of their immunological and biological effects, including methods for using BIOcore to determine binding kinetics and establishing various lymphocyte cell culture systems. There are also accounts of such methods as the RNase protection assay, cytokine ELISA, FACS analysis, and cytokine production at the single cell level..
MHC Protocols
The aim of MHC Protocols is to document protocols that can be used for the analysis of genetic variation within the human major histocompatibility complex (MHC; HLA region). The human MHC encompasses approximately 4 million base pairs on the short arm of chromosome 6 at cytogenetic location 6p21. 3. The region is divided into three subregions. The telomeric class I region contains the genes that encode the HLA class I molecules HLA-A, -B, and -C. The centromeric class II region contains the genes encoding the HLA class II molecules HLA-DR, -DQ, and -DP. In between is the class III region, originally identified because it contains genes encoding components of the complement pathway. The entir...
Lentivirus Gene Engineering Protocols
Cell gene engineering is emerging as a field with outstanding impact, not only in medicine/biology, but also, and perhaps most importantly, in agriculture and in all those food sciences involved in the fight against world hunger. Lentivirus vector-based technologies represent the last frontier in the development of powerful and reliable methods for both in vitro and in vivo gene transfer in eukaryotic animal cells. Although the design of lentivirus vectors is closely reminiscent of those already successfully applied to the construction of oncoretroviral vectors, some unique features, e.g., the ef- ciency in transducing both postmitotic and stem cells, render the use of lentivirus vectors inv...
Membrane Transporters
Studies of membrane transporters have had great impact on our und- standing human diseases and the design of effective drugs. About 30% of current clinically marketed drugs are targeting membrane transporters or channels. Membrane Transporters: Methods and Protocols provides various practical methodologies for the ongoing research on membrane transporters. To provide readers the most up-to-date information, several emerging fields and methodologies are embraced in this book, including pharmacogenomics, bioin-formatics, and microarray technology. Pharmacogenomics studies of membrane transporters are useful in drug discovery and in predicting drug responses in the clinic. In this volume, the c...
Recombinant Antibodies for Cancer Therapy
Since the advent of hybridoma technology more than two decades ago, numerous antibodies have entered the clinical setting as potent therapeutic agents. Their repeated application in humans, however, is limited by the development of human antimouse antibodies (HAMA) in the recipient, leading to allergic re- tions against the foreign murine protein and rapid neutralization. To circumvent these limitations many new antibodies have recently been tailored through recombinant antibody technology. The initial clinical data show encouraging results, thus demonstrating the potential of these new therapeutic agents. The purpose of Recombinant Antibodies for Cancer Therapy is to present a collection of...
E. coli Plasmid Vectors
A comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids, recombinant protein expression, and the use of reporter genes, are also described.
Capillary Electrophoresis of Carbohydrates
A collection of cutting-edge techniques for using capillary electrophoresis (CE) to analyze complex carbohydrates. These readily reproducible protocols provide methods for sample preparation, analysis of mono- and oligosaccharides, glycoproteins, and glycoconjugates. A useful appendix describes the structures of the most commonly encountered carbohydrate residues and olgosaccharides from mammalian and bacterial origins. Each protocol contains detailed information on reagents, apparatus, notes, comments, and tips on procedures.
Directed Enzyme Evolution
Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity c...
Protein Sequencing Protocols
Determination of the protein sequence is as important today as it was a half century ago, even though the techniques and purposes have changed over time. Mass spectrometry has continued its recent rapid development to find notable application in the characterization of small amounts of protein, for example, in the field of proteomics. The “traditional” chemical N-terminal sequencing is still of great value in quality assurance of the increasing number of biopharmaceuticals that are to be found in the clinic, checking processing events of recombinant proteins, and so on. It is joined in the armory of me- ods of protein analysis by such techniques as C-terminal sequencing and amino acid an...
