bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of Transfection
Authors: Rose John K. 
Published: June 2004

AbstractWhen embarking upon any transfection procedure, a critical first step is to optimize conditions. Every mammalian cell type has a characteristic set of requirements for optimal introduction of foreign DNA; there is a tremendous degree of variability in the transfection conditions that work, even among cell types that are very similar to one another. Often, an experimenter must screen a wide variety of cell types for a desired regulatory trait, such as an appropriate response to a particular effector molecule. It is thus helpful to have a straightforward, systematic approach to optimizing transfection efficiency. This unit provides guidelines to optimizing transfection experiments.
Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
Authors: Snapp Erik L. , Altan Nihal , Lippincott‐Schwartz Jennifer 
Published: June 2004

AbstractThis unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo‐induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.
Identification of Motifs in Protein Sequences
Authors: Sonnhammer Erik L.L. , Wolfsberg Tyra G. 
Published: June 2004

AbstractThis brief appendix serves as a guide for the analysis of functional motifs in proteins. Several database search engines that can be accessed via the World Wide Web are described. Such computerized searches have become the preferred method to scan large sequence and motif databases, as the searches are efficient and the databases are updated frequently. A short list of sorting signals is also included, since these motifs often cannot be predicted reliably by a computer search.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro 
Published: June 2004

AbstractThe use of radioisotopes to label specific molecules in a defined way has greatly advanced the discovery and dissection of biochemical pathways. The development of methods to inexpensively synthesize such tagged biological compounds on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie (mCi) amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries (mCi). In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the investigator. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses storage, handling, and disposal of 35S, 32P, 33P, and 125I.
Importing Biological Materials
Authors: Bryant Paula Wolf 
Published: June 2004

AbstractScientific cooperation is no longer confined to laboratories at the same institution, nor between laboratories within the boundaries of the United States. More than ever, scientists are forming collaborations with their peers outside the U.S., leading not only to the exchange of knowledge and ideas, but also to the exchange of biological reagents. Thus, investigators need to be aware that an import permit may be required to obtain purified proteins, chemically synthesized materials, cell lines, tissues, and other biological materials from abroad. Before requesting any biological material from a laboratory outside the U.S., it is the responsibility of the “receiver” to ascertain whether an import permit is required, to apply for the permit if one is determined to be necessary, and to provide the “shipper” with an official copy of the permit to be included with the shipped material. This unit identifies the appropriate agencies and steps required to obtain a permit.
Internet Basics for Biologists
Authors: Baxevanis Andreas D. , Ouellette B. F. Francis 
Published: June 2004

AbstractWith the explosion of sequence and structural information available to researchers, the field of bioinformatics is playing an increasingly large role in the study of fundamental biomedical problems. This information is generally available through the Internet and access to the World Wide Web. This unit provides an introduction to the internet–how it is organized, how you connect to it, using email and file transfer protocols, and finding your way around the Web.
Spectrophotometric Determination of Protein Concentration
Authors: Simonian Michael H. 
Published: June 2004

AbstractThis unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A280) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a280). Alternatively, absorbance measured at 205 nm (A205) is used to calculate the protein concentration. The A280 and A205 methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.
Dialysis and Concentration of Protein Solutions
Authors: Andrew Sarah. M. , Titus Julie. A. , Zumstein Louis 
Published: June 2004

AbstractConventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. Dialysis is usually used to change the salt (small‐molecule) composition of a macromolecule‐containing solution. This unit describes dialysis of large‐ and small‐volume samples using cellulose membranes with pore sizes designed to exclude molecules above a selected molecular weight. A support protocol describes preparation of membranes for dialysis and discusses issues related to the selection of membranes including commercial kits.
Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R. 
Published: June 2004

AbstractReliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in cell and molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques–Hoescht 33258 dye binding, ethidium bromide binding are described. These three procedures cover a range from 5 to 10 ng/ml DNA to 50m g/ml DNA.
Micro RT‐PCR
Authors: Paweletz Cloud P. , Charboneau Lu , Liotta Lance A. 
Published: June 2004

AbstractThis unit presents a collection of protocols for the microisolation, manipulation, and amplification of the RNA content of microdissected cells. Even though emphasis in these protocols is given for microdissected cells, these protocols have successfully been used for bulk tissue (i.e., less than 10 ug).
The Colorimetric Detection and Quantitation of Total Protein
Authors: Krohn Randall I. 
Published: June 2004

AbstractProtein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.
Searching Online Mendelian Inheritance in Man (OMIM) for Information for Genetic Loci Involved in Human Disease
Authors: Baxevanis Andreas D. 
Published: June 2004

AbstractOnline Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genomics researchers, physicians, and patients. OMIM is the electronic version of the catalog of human genes and genetic disorders. It provides concise textual information from the literature on most human conditions having a genetic basis, as well as pictures illustrating the condition or disorder (where appropriate) and full citation information. This unit gives an overview of the OMIM database, the layout of the records, and the information that is available within each entry.
Searching the NCBI Databases Using Entrez
Authors: Murphy Juliane , Baxevanis Andreas D. 
Published: June 2004

AbstractOne of the most widely‐used interfaces for the retrieval of information from biological databases is the NCBI Entrez system. Entrez capitalizes on the fact that there are pre‐existing, logical relationships between the individual entries found in numerous public databases. The existence of such natural connections, mostly biological in nature, argued for the development of a method through which all the information about a particular biological entity could be found without having to sequentially visit and query disparate databases. Two Basic Protocols describe simple, text‐based searches, illustrating the types of information that can be retrieved through the Entrez system. An builds upon the first Basic Protocol, using additional, built‐in features of the Entrez system, as well as providing alternative ways to issue the initial query. The reviews how to save frequently‐issued queries.
The UCSC Genome Browser
Authors: Karolchik Donna , Kent W. James 
Published: June 2004

AbstractThe UCSC Genome Browser is a popular Web‐based tool for quickly displaying a requested portion of a genome at any scale, accompanied by a series of aligned annotation tracks. The annotations ‐ some of which are generated at the University of California, Santa Cruz (UCSC) and some by external collaborators ‐ display gene predictions, mRNA alignments, expressed sequence tag alignments, cross‐species homologies, single nucleotide polymorphisms, and several other types of data. All information relevant to a region is presented in one window, facilitating biological analysis and interpretation. Users can also upload custom annotation tracks for research or educational use. This unit describes how to use and customize the UCSC Genome Browser, download the underlying database tables, and create and display custom annotation tracks.
Using the TIGR Gene Index Databases for Biological Discovery
Authors: Lee Yuandan , Quackenbush John 
Published: June 2004

AbstractThe TIGR Gene Index web pages provide access to analyses of ESTs and gene sequences for nearly 60 species, as well as a number of resources derived from these. Each species‐specific database is presented using a common format with a homepage. A variety of methods exist that allow users to search each species‐specific database. Methods implemented currently include nucleotide or protein sequence queries using WU‐BLAST, text‐based searches using various sequence identifiers, searches by gene, tissue and library name, and searches using functional classes through Gene Ontology assignments. This protocol provides guidance for using the Gene Index Databases to extract information.
Searching the Mouse Genome Informatics (MGI) Resources for Information on Mouse Biology from Genotype to Phenotype
Authors: Shaw David 
Published: June 2004

AbstractThe Mouse Genome Informatics (MGI, http://www.informatics.jax.org/) resource provides the research community with access to information on the genetics, genomics, and biology of the laboratory mouse. Core data in MGI include gene characterization and function, phenotype and disease model descriptions, DNA and protein sequence data, gene expression data, mammalian orthologies, polymorphisms, mapping data, and links to other bioinformatics databases. Semantic integration is supported through the use of standardized nomenclature and controlled vocabularies such as the mouse Anatomical Dictionary and the Gene Ontologies. MouseBLAST offers a fast, sequence‐based method for identifying genes and searching for similarity, and provides links from sequence to MGI biological data. MGI extracts and organizes data from primary literature. MGI data are shared with and widely displayed by other bioinformatics resources. The database is updated nightly with curated annotations and regularly adds new datasets and enhances user interface features. This unit provides a guide to using the MGI bioinformatics resource.
An Introduction to Recognizing Functional Domains
Authors: Stormo Gary 
Published: June 2004

AbstractThis unit provides an overview of issues involved in domain recognition in protein and DNA sequences. It opens with a discussion of the two primary methods of domain representation, namely consensus sequences and alignment matrices (e.g., the log‐odds matrix). The unit continues with a brief overview of some of the resources available for identifying functional domains in nucleotide sequences (e.g., TRANSFAC, TESS, and MEME). In addition, it reviews databases such as Pfam, and Blocks, which are available for protein analysis.
Multiple Sequence Alignment Using ClustalW and ClustalX
Authors: Thompson Julie D. , Gibson Toby. J. , Higgins Des G. 
Published: June 2004

AbstractThe Clustal programs are widely used for carrying out automatic multiple alignment of nucleotide or amino acid sequences. The most familiar version is ClustalW, which uses a simple text menu system that is portable to more or less all computer systems. ClustalX features a graphical user interface and some powerful graphical utilities for aiding the interpretation of alignments and is the preferred version for interactive usage. Users may run Clustal remotely from several sites using the Web or the programs may be downloaded and run locally on PCs, Macintosh, or Unix computers. The protocols in this unit discuss how to use ClustalX and ClustalW to construct an alignment, and create profile alignments by merging existing alignments.
Discovering Novel Sequence Motifs with MEME
Authors: Bailey Timothy L. 
Published: June 2004

AbstractThis unit illustrates how to use MEME to discover motifs in a group of related nucleotide or peptide sequences. A MEME motif is a sequence pattern that occurs repeatedly in one or more sequences in the input group. MEME can be used to discover novel patterns because it bases its discoveries only on the input sequences, not on any prior knowledge (such as databases of known motifs). The input to MEME is a set of unaligned sequences of the same type (peptide or nucleotide). For each motif it discovers, MEME reports the occurrences (sites), consensus sequence, and the level of conservation (information content) at each position in the pattern. MEME also produces block diagrams showing where all of the discovered motifs occur in the training set sequences. MEME's hypertext (HTML) output also contains buttons that allow for the convenient use of the motifs in other searches.
Using TESS to Predict Transcription Factor Binding Sites in DNA Sequence
Authors: Schug Jonathan 
Published: June 2004

AbstractThis unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence‐specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.
The InterPro Database and Tools for Protein Domain Analysis
Authors: Mulder Nicola J. , Apweiler Rolf 
Published: June 2004

AbstractInterPro provides a one‐stop shop for protein‐sequence classification, freeing the user from having to visit multiple database separately and rationalize the different results in varying formats. This unit describes how to submit a sequence to InterProScan via a Web or E‐mail server. It also provides instructions for installing and running InterProScan locally. In addition, details on browsing InterPro families and domains of interest using the InterPro Web and sequence retrieval system (SRS) are provided.
An Overview of Sequence Similarity (“Homology”) Searching
Authors: Davison Daniel B. 
Published: June 2004

AbstractSequence similarity searching is an essential tool for molecular biologists. It is used to support inference of protein function and for phylogenetic analysis. Every searching procedure requires some understanding of the underlying principles, so at the very least the investigator's selection of parameters is correct. The principles underlying the most commonly used procedures are presented in this unit. The discussion is intentionally non‐mathematical, but does contain references for those who desire a mathematical and statistical discussion of these procedures.
Finding Homologs to Nucleotide Sequences Using Network BLAST Searches
Authors: Ladunga Istvan 
Published: June 2004

AbstractBasic Local Alignment Search Tool (BLAST) can identify possible homologs in nucleotide and protein databases with high sensitivity and selectivity, and at an amazing speed. Those homologs may provide inference for the biochemical function, the exon boundaries, the domain architecture, the secondary and tertiary structure of the protein, and many other features. The purpose of this unit is not restricted to providing optimal ways of applying the BLAST tool. Just running a BLAST search is an easy task and still can be of great service to researchers. The unit details how to fine tune the arguments of the programs, allowing the user to take advantage of frequently overlooked capabilities of the tool. It also addresses the pitfalls in over‐ or under‐interpretation of results.
Constructing and Refining Multiple Sequence Alignments with PileUp, SeqLab, and the GCG Suite
Authors: Thompson Steven M. 
Published: June 2004

AbstractThis unit discusses how the Accelrys GCG Wisconsin Package SeqLab graphical user interface can be used to align, annotate, analyze, and export into alternative formats, multiple biological sequence data. The emphasis is on discovering and recognizing common elements within the dataset. The GCG programs, or implementations of public domain programs thereof, investigated include: LookUp, PileUp, PlotSimilarity, FASTA, Motifs, MEME/MotifSearch, the Profile Package, the HMMER Package, PAUPSearch, and ToFastA. ReadSeq, a non‐GCG, public domain program is also used.
Computing Multiple Sequence/Structure Alignments with theT‐Coffee Package
Authors: Notredame Cedric , Suhre Karsten 
Published: June 2004

AbstractThe FASTA package provides a comprehensive set of similarity searching programs, similar to those provided by the BLAST package, and some additional programs for searching with short peptides and oligonucleotides that are not provided by BLAST. The FASTA programs work with a wide variety of database formats, including mySQL sequence databases. FASTA provides very accurate statistical significance estimates, and is more sensitive than BLASTN when comparing DNA sequences. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons.
Finding Protein and Nucleotide Similarities with FASTA
Authors: Pearson William 
Published: June 2004

AbstractThe FASTA package provides a comprehensive set of similarity searching programs, similar to those provided by the BLAST package, and some additional programs that are not provided by BLAST for searching with short peptides and oligonucleotides. The FASTA programs work with a wide variety of database formats, including mySQL sequence databases. FASTA provides very accurate statistical significance estimates, and is more sensitive than BLASTN when comparing DNA sequences. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons.
Mathematically Complete Nucleotide and Protein Sequence Searching Using Ssearch
Authors: Ropelewski Alexander J. , Nicholas Hugh B. , Deerfield David W. 
Published: June 2004

AbstractIn this unit a protocol is described for predicting the structure of simple transmembrane a‐helical bundles. The protocol is based on a global molecular dynamics search (GMDS) of the configuration space of the helical bundle, yielding several candidate structures. The correct structure among these candidates is selected using information from silent amino acid substitutions, employing the premise that only the correct structure must (by definition) accept all of the silent amino acid substitutions. Thus, the correct structure is found by repeating the GMDS for several close homologs and selecting the structure that persists in all of the trials.
Installing, Maintaining, and Using a Local Copy of BLAST for Intranet and Workstation Use
Authors: Littlejohn Timothy G. 
Published: June 2004

AbstractThe Basic Local Alignment Search Tool (BLAST) is one of the widest used and most useful applications in sequence‐based bioinformatics analysis. Frequently it is not practical or possible to use remote BLAST services through the Internet due to restrictions of a security or technical nature or the need for high‐throughput analysis requiring greater amounts of processing power than are available from remote services. This unit describes the steps involved in obtaining and installing a copy of the BLAST software for use on a local intranet or stand‐alone workstation. Once installed, the BLAST package can be used to create BLAST‐searchable nucleotide and protein sequence databanks. Various popular hardware (PPC, Intel) and operating system (MacOSX, FreeBSD and Linux) options for running and maintaining the software are discussed. Finally, steps for indexing proprietary and third party (publicly available) sequence databanks for use with BLAST and managing these resources are discussed.