Showing papers in "Methods in 2002"

TL;DR: Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible and here a collection of protocols for siRNA-mediated knockdown of mammalian gene expression is provided.

TL;DR: Reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids are described.

TL;DR: The complete way to characterize an UV source is by spectroradiometry, although for most practical purposes a detector optically filtered to respond to a limited portion of the UV spectrum normally suffices.

TL;DR: The results indicate that the RIP assay is a powerful tool to identify RNA-protein interactions in vivo and has the potential to unravel the cellular network of RNP complexes in their native setting.

TL;DR: How to set up the basic system and outline modifications that can be applied to address specific research questions are reviewed and new ways of identifying protein spots in Blue Native gels using mass spectrometry are discussed.

TL;DR: Two modifications to the standard chromatin immunoprecipitation protocol are reviewed, designed to identify novel target genes of transcription factors in mammalian cells and are ideal for a more global analysis of target genes.

TL;DR: This article focuses on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrates the necessity of complete protein removal from DNA samples prior to bisulfITE treatment.

TL;DR: It is hypothesized that mRNAs are organized into structurally and functionally linked groups to better affect information transfer through coordinate gene expression to facilitate the production of proteins that regulate processes necessary for growth and differentiation.

TL;DR: A set of accurate polarographic and spectrophotometric assays that use relatively small amounts of biological material and discuss the biochemical parameters appropriate for discriminating partial OXPHOS defects are described.

TL;DR: Weinmann and Farnham as discussed by the authors described complementary methods for detailed in vivo characterizations of such identified protein-DNA interactions, including formaldehyde crosslinking and chromatin immunoprecipitation.

TL;DR: This article describes a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues and an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues.

TL;DR: The selection of an appropriate AAV vector for ocular gene transfer studies is described and the techniques used to deliver the virus to the eye and to assess ocular transfection are discussed.

TL;DR: This article examines recent methods for measuring reactive oxygen species produced in isolated mitochondria and within live cells, with particular emphasis on the detection of hydrogen peroxide, and provides advice to aid the interpretation of cellular data.

TL;DR: The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation.

TL;DR: The physical principles on which BIACORE is based, and the required instrumentation, are discussed, and how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data are described.

TL;DR: A three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae can be described and used to detect or confirm an RNA- Protein interaction, to analyze RNA- protein interactions genetically, and to discover new protein or RNA partners when only one is known.

TL;DR: Evaluated UVR-induced erythema in previously unexposed buttock skin of volunteers of skin types I, II, III, and IV showed that there is a trend for increased SSR MED with skin type, with the MED of skin type IV being approximately twice that of skintype I, a smaller difference than one might have expected.

TL;DR: This review summarizes and compares different promoters and regulatory elements that have been used with r AAV as a reference toward achieving a high level of rAAV-mediated transgene expression in the CNS.

TL;DR: It is recommended that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.

TL;DR: The central focus of this review is the use of ChIP to study transcriptional activation over long distances on chromosomes.

TL;DR: There is a need to validate recombinant proteins to ascertain equivalence with their native counterparts when used in immunological studies, diagnostics, and immunotherapy as interest in recombinant latex allergens increases.

TL;DR: Two strategies are compared that rely on transfection of cells in culture with either two or three plasmids, containing the AAV vector genome and encoding AAV and adenoviral helper functions, and possible further improvements proposed.

TL;DR: No universally "best" method is applicable to all neurotransporter imaging studies, and careful evaluation of model-based methods is required for each radiotracer.

TL;DR: A review of biochemical and structural approaches to characterization of restriction endonucleases can be found in this article, with particular attention to the multiple crucial roles of divalent metal ions, the possibilities for use of alternative substrates in binding and catalytic experiments, and the strategies for exploring the detailed chemistry of phosphoryl transfer.

TL;DR: This work exploits chemostat culture, in which the cells can be grown at a fixed growth rate, in combination with hybridization array technology, to investigate the effect of carbon and nitrogen limitation at the transcriptome level.

TL;DR: Although this experimental system is quite far removed from the epigenetic controls acting during development it does provide the means to clarify the rules governing the silencing of genes by specific DNA methylation and their reactivation by demethylation, which will facilitate studies on the control of gene expression in somatic cells of the developing organism or the adult.

TL;DR: A method to determine the extent of cytosine methylation in DNA is described, which gives highly reproducible results and is capable of measuring 5-methylcytosine levels in as little as 1 microg of DNA.

TL;DR: A detailed protocol for purification of native Cre is outlined and straightforward assays that can be used to test for recombination activity in vitro are described and should be useful to any investigator with a need for purified Cre recombinase.

TL;DR: The biochemical and biophysical methods involved in the detection of receptor dimers are described and some of the concerns are addressed and precautions to be taken are suggested in studies examining receptor-receptor interactions.

TL;DR: Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent identification of these proteins by mass spectrometry, which led to the identification of three novel subunits.