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Extraction of Chondroitin/Dermatan Sulfate Glycosaminoglycans from Connective Tissue for Mass Spectrometric AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) are present in high levels in connective tissue where they play roles as structural molecules and in protein-binding interactions. Recent developments in the techniques for analysis of CS/DS using capillary electrophoresis (CE) have enabled progress in the understanding of changes in CS/DS structure that accompany connective tissue diseases including osteoarthritis. Key to these developments is the ability to extract CS/DS GAGs from small quantities of connective tissue. This chapter describes a method for connective tissue GAG extraction, derivatization, and w...
Source: Springer protocols feed by Biochemistry - October 30, 2009 Category: Biochemistry Source Type: info

A Glycomics Approach to the Discovery of Potential Cancer BiomarkersEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Glycosylation is highly sensitive to the biochemical environment and plays a key role in development and disease manifestation. Moreover, glycan biosynthesis depends on several highly competitive processes; thus, variations in the concentration of specific glycosyltransferases produce different products. For this reason, monitoring changes in glycosylation may be a more specific and sensitive approach to biomarker discovery and possibly disease diagnosis. Glycans in serum are of particular interest as approximately half of all proteins are glycosylated. We have developed the methods for profiling the glycans in human serum...
Source: Springer protocols feed by Biochemistry - October 30, 2009 Category: Biochemistry Source Type: info

Characterization of Lipid-Linked Oligosaccharides by Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
N- Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes until the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. We have developed a new glycomics strategy based on lectin-affinity capture of lipid-linked oligosaccharides (LLOs) coupled to capillary electrophoresis mass spectrometry. The LLO intermediates of the C. jejuni Pgl pathway were used to validate the methodology and to better characterize the bacterial model system for protein N-glycosylation. ...
Source: Springer protocols feed by Biochemistry - October 30, 2009 Category: Biochemistry Source Type: info

Metabolomics in GlycomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Metabolomics is essentially the study of all low molecular weight molecules in a biological system under defined conditions. In glycomics, there is much potential to gain insight into the biosynthesis of novel glycoconjugate structures by probing the metabolome for substrates that are suspected, or known, to be involved in the biosynthetic processes. Recently, we employed the use of hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) in a focused metabolomic study of sugar-nucleotides relevant to the biosynthesis of highly novel carbohydrate modifications on the flagellin of Campylobacter...
Source: Springer protocols feed by Biochemistry - October 30, 2009 Category: Biochemistry Source Type: info

The Application of NMR Spectroscopy to Functional GlycomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis ...
Source: Springer protocols feed by Biochemistry - October 30, 2009 Category: Biochemistry Source Type: info

Microarray-Based Study of Carbohydrate–Protein BindingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To develop a novel high-throughput tool for monitoring carbohydrate–protein interactions, carbohydrate or glycoprotein microarrays have been prepared for binding with lectins. The interaction events are marked by attachment of fluorescent dyes and gold nanoparticles. The attachment of the fluorescent dyes and gold nanoparticles is achieved by standard avidin–biotin chemistry. The detection principle is fluorescence or resonance light scattering (RLS). The electroless deposition of silver onto the gold particles has been employed for RLS signal enhancement. Well-defined recognition systems, three monosaccharides...
Source: Springer protocols feed by Biochemistry - October 30, 2009 Category: Biochemistry Source Type: info

In Toto Imaging of Embryogenesis with Confocal Time-Lapse MicroscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microscopy has been one of the most direct and powerful tools since the beginning of biological research. Continued advances such as confocal and two-photon fluorescence microscopy and fluorescent proteins now make imaging useful at a variety of spatial scales (molecules, circuits, cells, tissues, and even whole embryos) and temporal scales (<seconds to days). Zebrafish is uniquely poised to benefit from these continued technological improvements because of its inherent suitability for both imaging and genetics. This chapter presents an approach called “in toto imaging”. The goal of in toto imaging is to ima...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Morphologic Analysis of the Zebrafish Digestive SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Imaging Zebrafish Embryos by Two-Photon Excitation Time-Lapse MicroscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. In particular, 2PE microscopy has been shown to be useful for imaging deep cell layers within the embryo and following tissue morphogenesis over long periods. This chapter describes how to use 2PE microscopy to study morphogenetic movements during early zebrafish embryonic development, providing a general blueprint for its use...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Live Cell Imaging of Zebrafish LeukocytesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Zebrafish are ideally suited for the live imaging of early immune cell compartments. Macrophages that initially appear on the yolk surface prior to the onset of circulation are the first functional immune cells within the embryo, predating the emergence of the first granulocytic cells—the heterophilic neutrophils. Both cell types have been shown in zebrafish to contribute to a robust early innate immune system, capable of clearing systemic infections and participating in wound healing. Early imaging of these cells within zebrafish relied on differential interference contrast (DIC) optics because of their superficial ...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Multiple Embryo Time-Lapse Imaging of Zebrafish DevelopmentEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Understanding the dynamics of developmental and cellular processes requires documentation of their changes with appropriate temporal and spatial resolution. Furthermore, simultaneous recording from a population of embryos under identical conditions allows statistical estimates of precision and variability to be made. This chapter describes a protocol for time-lapse microscopy of multiple embryos in parallel developing under tightly controlled conditions. This method is currently best suited to follow tissue-scale morphogenetic movements with temporal resolution in the minute range, for hours or even days. Applications of t...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Validating microRNA Target Transcripts Using Zebrafish AssaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a method for a reporter assay based on a fluorescence intensity readout that uses transient techniques in zebrafish to easily deliver the reporter assay components. In addition, we describe a rigorously controlled strategy for determining the bona fide miRNA binding sites in the 3′UTR of mRNAs. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Zebrafish Spotted-Microarray for Genome-Wide Expression Profiling Experiments: Data Acquisition and AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In expression microarray experiments, post-hybridization procedures involving data acquisition and analysis are mainly computational and hence are described as “dry-lab procedures.” The aim of these procedures is to acquire the fluorescent signals representing the relative abundance of tens of thousands of RNA species in samples with biological interest, and transform them into meaningful data with biological significance. These procedures begin with image and data acquisition followed by data normalization and processing, and thereafter primary and secondary data analyses. Although the actual data analysis is ...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Zebrafish Spotted-Microarray for Genome-Wide Expression Profiling Experiments. Part I: Array Printing and HybridizationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The availability of microarray technology for zebrafish research has enabled the expression of tens of thousands of genes to be studied simultaneously in one experiment. The experiment usually involves measuring and comparing the relative abundance of tens of thousands of mRNA species in experimental samples obtained from mutant versus wild-type embryos, disease versus normal tissues, embryos/fish of different developmental stages, physiologic states, or from multiple treatments and/or time-points. A microarray experiment comprised of several stages can be divided into two distinct parts (i.e., the “wet-lab” an...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Nitroreductase-Mediated Cell Ablation in Transgenic Zebrafish EmbryosEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe transgenic methods to express the Escherichia coli nfsB in a tissue restricted manner in the zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme that can render prodrugs such as metronidazole (Met) cytotoxic. Using the expression of NTR fused to a fluorescent protein, one can simultaneously make cells susceptible to prodrug treatment and visualize cell ablation as it occurs. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Tissue Micromanipulation in Zebrafish EmbryosEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although a common approach in large vertebrate embryos such as chick or frog, manipulation at the tissue level is only rarely applied to zebrafish embryos. Despite its relatively small size, the zebrafish embryo can be readily used for micromanipulations such as tissue and organ primordium transplantation, explantation, and microbead implantation, to study inductive tissue interactions and tissue autonomy of pleiotropic, mutant phenotypes or to isolate tissue for organotypic and primary cell culture or RNA isolation. Since this requires special handling techniques, tools, and tricks, which are rarely published and thus dif...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Focal Electroporation in Zebrafish Embryos and LarvaeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A method is described that allows the introduction by electroporation of either small dyes or larger RNA, DNA, or morpholino constructs into single cells or small groups of cells in zebrafish embryos or larvae. The dye or construct is delivered to cells via a patch-like microelectrode that also delivers the electroporation stimulus train. This technique allows the experimenter to target cells of their choice at a particular time of development and at a particular location in the embryo, and is useful for fate mapping, analysing neuronal organisation, ectopic expression and gene knockdown experiments. (Source: Springer prot...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Simple and Efficient Transgenesis with Meganuclease Constructs in ZebrafishEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Bacterial Artificial Chromosome Transgenesis for ZebrafishEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transgenesis using bacterial artificial chromosomes (BAC) allows greater fidelity in directing desirable expression of transgenes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of gene expression in a living organism. We have developed a streamlined procedure of directly selecting multiple BAC clones based on public sequence databases followed by rapid modification with green fluorescent protein or red fluorescent protein for transgenic analysis in zebrafish. In this chapter, experimental procedures for BAC DNA preparation and g...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Analysis of Genes and Genome by the Tol2-Mediated Gene and Enhancer Trap MethodsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene trap and enhancer trap methods have been developed. The gene trap and enhancer trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 transcription activator gene. By creating random integrations of these constructs in the genome, transgenic fish expressing the GFP gene or the Gal4 gene in specific cells, tissues or organs are generated. These fish are valuable resources for developmental biology. Especially, the Gal4-expressing transgenic fish can be used to ectopically ...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Transient and Stable Transgenesis Using Tol2 Transposon VectorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transgenesis is an important methodology for studying the function of genes and genomes in model plants and animals. For the model vertebrate zebrafish, methods using the Tol2 transposable element have been developed for this purpose. With these methods, the function of the transgene can be analyzed in both transient and stable transgenic fish. Recently, cis-sequences necessary for transposition of the Tol2 element were revealed. This enabled development of transposon vectors containing minimal DNA sequences that are easily manipulated. More recently, several transposon vectors containing the Gateway sequence were created ...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Cryopreservation and In Vitro Fertilization at the Zebrafish International Resource CenterEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In recent decades, laboratories throughout the world generated several thousand mutant, transgenic, and wild-type zebrafish lines and more lines continue to be produced. At the same time, relatively little effort has been expended to develop reliable, high-throughput, standardized, long-term cryopreservation storage methods, even though laboratories and the research community as a whole struggle to maintain the large number of lines alive. Safe and reliable methods for maintaining these valuable genetic resources are vital for future biomedical research. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Undertaking a Successful Gynogenetic Haploid Screen in ZebrafishEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chemical mutagenesis using N-ethyl-N-nitrosourea is the current method of choice for dense mutagenesis in zebrafish. Methods are available for both pre-meiotic and post-meiotic sperm mutagenesis; in this chapter, pre-meiotic mutagenesis is described. Mutated males are crossed with untreated females to create an F1 generation that is heterozygous for the mutations. The F1 females can be screened directly by making haploid embryos using in vitro fertilization (IVF) with ultraviolet (UV)-irradiated sperm. This approach requires substantially fewer fish and less aquarium space than the classical F2 generation screen and is fea...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Production of Pseudotyped Retrovirus and the Generation of Proviral Transgenic ZebrafishEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a quantitative PCR (qPCR)-based assay as a quick way to assess the infectivity after each round of viral production and injection. Most of the required equipment is commercially available and commonly present in most research laboratories. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Highly Efficient ENU Mutagenesis in ZebrafishEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and reverse genetic approaches. To obtain high-density mutagenized zebrafish, six consecutive ENU treatments are applied at weekly intervals to adult male zebrafish by bathing them in ENU solution. With this procedure an average germ line mutation load of one mutation every 1.0 × 105–1.5 × 105 basepairs is rea...
Source: Springer protocols feed by Biochemistry - August 20, 2009 Category: Biochemistry Source Type: info

Nanopore Force Spectroscopy on DNA DuplexesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present the details of the measurement of the duplex survival probability under force, and show that dissociation time scales for duplexes that are perfectly complimentary differ by greater than approximately two orders of magnitude from those containing a single sequence mismatch, offering opportunities for sequence detection. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Microfluidic Devices with Photodefinable Pseudo-valves for Protein SeparationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plastic microfluidic devices are fabricated with an array of pseudo-valves for two-dimensional (2D) protein separation. The devices are made by compression molding; the mold is created by electroplating on a glass master fabricated by photolithography. Each device consists of one channel for isoelectric focusing (IEF) and multiple parallel channels for polyacrylamide gel electrophoresis (PAGE). The IEF channel (first dimension) is orthogonal to the PAGE channels (second dimension). Microfluidic pseudo-valves are created at the intersections of orthogonal channels by photodefinable, in situ gel polymerization. These valves ...
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Formulation/Preparation of Functionalized Nanoparticles for In Vivo Targeted Drug DeliveryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Targeted cancer therapy allows the delivery of therapeutic agents to cancer cells without incurring undesirable side effects on the neighboring healthy tissues. Over the past decade, there has been an increasing interest in the development of advanced cancer therapeutics using targeted nanoparticles. Here we describe the preparation of drug-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(d,l-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2-fluoropyrimidine ribonucleic acid aptamers that recognize the extracellular domain of...
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Detection of mRNA in Single Living Cells Using AFM NanoprobesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Examining messenger RNA (mRNA) expression is useful for the determination of cell and tissue conditions. Many methods of determining mRNA expression require total RNA extraction or cell fixation. These processes cause difficulties in examining mRNA expression in single living cells without causing cell death. We think that analysis of specific mRNA expression in single living cells will become important in cell biology. In this chapter, we present a method to examine mRNA expression of single living cells without killing the cells. The single-cell nanoprobe (SCN) method uses atomic force microscopy (AFM) to extract mRNA. W...
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian GenomesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to...
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Reverse Transfection Using Gold NanoparticlesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a method for reverse transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagentcomplex on a glass surface, which could then be used for the regulation of the particle size of the complex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet–PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared with the control w...
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Determining DNA Sequence Specificity of Natural and Artificial Transcription Factors by Cognate Site Identifier AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Artificial transcription factors (ATFs) are designed to mimic natural transcription factors in the control of gene expression and are comprised of domains for DNA binding and gene regulation. ATF domains are modular, interchangeable, and can be composed of protein-based or nonpeptidic moieties, yielding DNA-interacting regulatory molecules that can either activate or inhibit transcription. Sequence-specific targeting is a key determinant in ATF activity, and DNA-binding domains such as natural zinc fingers and synthetic polyamides have emerged as useful DNA targeting molecules. Defining the comprehensive DNA binding specif...
Source: Springer protocols feed by Biochemistry - May 31, 2009 Category: Biochemistry Source Type: info

Use of Bimolecular Fluorescence Complementation in Yeast Saccharomyces cerevisiaeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Visualization of protein–protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. Bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as “tags” to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for use of BiFC in the yeast Saccharomyces cerevisiae. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Genome-Wide Analysis of Membrane Transport Using Yeast Knockout ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The transport of membrane-bound proteins through post-Golgi compartments depends on the coordinated function of multiple genes that direct the recognition and routing of protein cargoes to their final cellular destination. As many of these sorting components are nonessential for viability, genome-wide screening of the yeast gene-deletion mutant collection provides a useful strategy for their identification. The potential of this approach is limited only by the availability of transport assays suitable for the high-throughput screening of yeast colony arrays. Two large-scale phenotypic screens to identify novel transport ge...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

High-Throughput Protein Extraction and Immunoblotting Analysis in Saccharomyces cerevisiaeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A variety of Saccharomyces cerevisiae strain libraries allow for systematic analysis of strains bearing gene deletions, repressible genes, overexpressed genes, or modified genes on a genome-wide scale. Here we introduce a method for culturing yeast strains in 96-well format to achieve log-phase growth and a high-throughput technique for generating whole-cell protein extracts from these cultures using sodium dodecyl sulfate and heat lysis. We subsequently describe a procedure to analyze these whole-cell extracts by immunoblotting for alkaline phosphatase and carboxypeptidase yscS to identify strains with defects in protein ...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

A Cell-Free System for Reconstitution of Transport Between Prevacuolar Compartments and Vacuoles in Saccharomyces cerevisiaeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Genetic approaches have revealed more than 50 genes involved in the delivery of soluble zymogens like carboxypeptidase Y (CPY) to the lysosome-like vacuole in Saccharomyces cerevisiae. At least 20 of these genes function in transport between the prevacuolar endosome-like compartment (PVC) and the vacuole. To gain biochemical access to these functions, the authors developed a cell-free assay that measures transport-coupled proteolytic maturation of soluble zymogens in vitro. A polycarbonate filter with a defined pore size is used to lyse yeast spheroplasts after pulse-chase radiolabeling. Differential centrifugation enriche...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

In vitro Analysis of the Mitochondrial Preprotein Import Machinery Using Recombinant Precursor PolypeptidesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The import of precursor proteins into mitochondria represents a cell biological process that is absolutely required for the survival of an eukaryotic cell. A complex chain of reactions needs to be followed to achieve a successful transport of mitochondrial proteins from the cytosol through the double membrane system to their final destination. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Although most of...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

In Vitro Import of Proteins Into Isolated MitochondriaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Import of proteins is of vital importance for the biogenesis of mitochondria. The vast majority of mitochondrial proteins is encoded within the nuclear genome and translocated into various mitochondrial compartments after translation in the cytosol as preproteins. Even in rather primitive eukaryotes like yeasts, these are 700 to 1,000 different proteins, whereas only a handful of proteins is encoded in the mitochondrial DNA. In vitro import studies are important tools to understand import mechanisms and pathways. Using isolated mitochondria and radioactively labeled precursor proteins, it was possible to identify several i...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

In Vivo Labeling and Analysis of Mitochondrial Translation Products in Budding and in Fission YeastsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mitochondrial biogenesis requires the contribution of two genomes and of two compartmentalized protein synthesis systems (nuclear and mitochondrial). Mitochondrial protein synthesis is unique on many respects, including the use of a genetic code with deviations from the universal code, the use of a restricted number of transfer RNAs, and because of the large number of nuclear encoded factors involved in assembly of the mitochondrial biosynthetic apparatus. The mitochondrial biosynthetic apparatus is involved in the actual synthesis of a handful of proteins encoded in the mitochondrial DNA. The budding yeast Saccharomyces c...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Synthesis and Sorting of Mitochondrial Translation ProductsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mitochondria are essential organelles of eukaryotic cells. The biogenesis of mitochondria depends on the coordinated function of two separate genetic systems: one in the nucleus and one in the organelle. The study of mitochondria requires the analysis of both genetic systems and their protein products. In this chapter, we focus on the translation and sorting of mitochondrially encoded proteins into the mitochondrial inner membrane in the baker’s yeast Saccharomyces cerevisiae. The starting point is the labeling of these proteins, followed by some of the methods developed to investigate their topology and membrane inc...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Exploring Protein–Protein Interactions Involving Newly Synthesized Mitochondrial DNA-Encoded ProteinsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Biogenesis of the mitochondrial respiratory chain enzymes involves the coordinated action of the mitochondrial and nuclear genomes. As a matter of fact, the structural subunits forming these multimeric enzymes are encoded in both genomes. In addition, the assistance of nuclear encoded factors, termed assembly factors, is necessary to allow for the expression of the mitochondrial DNA-encoded subunits and to facilitate their maturation, membrane insertion, and further assembly into the corresponding enzymatic complex. These processes involve transient interactions among the newly synthesized mitochondrial products and specif...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Using Quantitative Fluorescence Microscopy to Probe Organelle Assembly and Membrane TraffickingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present here an overview of the principles involved in doing quantitative fluorescence microscopy. We illustrate these with examples drawn from our work with the Golgi apparatus and endosomes in cultured mammalian cells. The principles themselves can be applied to any system. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Measuring Secretory Membrane Traffic: A Quantitative Fluorescence Microscopy ApproachEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this chapter the authors describe automated imaging methods to quantify the transport rates of transmembrane as well as soluble cargo, and to evaluate the integrity of the Golgi complex. The quantification of cargo transport rates serves as an example of fluorescence intensity-based assays, the quantification of the Golgi complex integrity—as an example of morphology-based assays. These quantitative assays could be applied for single experiments as well as for middle- and high-throughput screening approaches. Each of these assays can be used to appreciate effects caused by gene silencing by RNAi, cDNA overexpressi...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

A Correlative Light and Electron Microscopy Method Based on Laser Micropatterning and EtchingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Correlative microscopy is a hybrid method that allows the localization of events observed under visible, ultraviolet, or infrared light, at molecular and submolecular levels, combining two microscopy techniques. However, the main limitation of correlative microscopy is to develop a labeling technique that can be easily used first in light and then in electron microscopy. Laser etching is a well-established method to create precisely designed shapes or volumes in various materials including glass. We have applied this technique to develop a new correlative light and electron microscopy method and to apply it in our study of...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Recruitment of Coat Proteins to PeptidoliposomesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Intracellular transport between compartments within the cell is generally mediated by membrane vesicles. Their formation is initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo proteins, and deform the lipid bilayer. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. To analyze the involvement of membrane proteins, the cytosolically exposed sequences may be coupled chemically to reactive lipids in the membrane. Here we describe the use of such peptidoliposomes presenting lipid-coupled cytoso...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Approaches to Investigate the Role of Signaling in ER-to-Golgi TransportEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
There have been many indications that kinases play a role in signaling the transport of proteins through the secretory pathway. Specifically, the serine/threonine kinase Akt affects the transport of cholesterol regulatory components from the endoplasmic reticulum (ER) to the Golgi. However, elucidating the target of Akt in this process has proven to be challenging. Here we describe an approach devised to investigate the Akt target(s) based on examining a potential candidate. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

SNARE-Mediated Fusion of LIposomesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lipid-mixing assay is now commonly used to study protein, temperature and ion-dependent membrane fusion events. This assay has been crucial to demonstrate the ability of neuronal and non-neuronal soluble NSF attachment receptor (SNARE) to promote spontaneous fusion of liposomes. This lipid-mixing assay is based on the fluorescence resonance energy transfer (FRET) capability between a donor fluorescent lipid and a quenching lipid. When fusion between donor fluorescent liposomes and nonfluorescent acceptor liposome occurred, FRET decreases. This assay allows a real-time reading of SNARE-mediated liposome fusion. (Source: Spr...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Use of Polarized PC12 Cells to Monitor Protein Localization in the Early Biosynthetic PathwayEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi appar...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Tracking the Transport of E-Cadherin to and From the Plasma MembraneEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The epithelial to mesenchymal transition (EMT) is the breakdown of epithelial cell morphology that gives way to a more mobile, mesenchymal phenotype. Although this process is fundamental to the development of multicellular organisms, it is also a key occurrence in many diseases, including cancers of epithelial origin E-cadherin is a central component of adherens junctions (AJs), which act as structural and signaling hubs in epithelial cells that oppose EMT. The loss of E-cadherin from the plasma membrane is an early indication of EMT and a marker of poor prognosis in many cancers making the trafficking of E-cadherin an are...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info

Analysis of Nucleocytoplasmic Shuttling of NFκB Proteins in Human LeukocytesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Controlled nucleocytoplasmic localization regulates activity of NFκB as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NFκB activity in human neutrophils by increasing the nuclear accumulation of NFκB inhibitor, IκBα. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of IκBα in human macrophage-like U937 cells, thus inhibiting N...
Source: Springer protocols feed by Biochemistry - June 6, 2008 Category: Biochemistry Source Type: info