Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.The Past, Present, and Future of Chemical Risk Assessment
Chemicals are essential components of our everyday life, from industrial chemicals used for manufacturing processes to the active principles of pharmaceuticals. As a consequence, whether it is accidental or deliberate, the population is now acutely and chronically exposed to chemicals, which has strengthened the need for improved methods of chemical risk assessment. Decades of experience with animal-based toxicity testing strategies have shown that their collective prediction of human risk is not satisfactory. Thus there is now a focus on developing human-based in vitro models with the goal to overcome this predictivity ga...
Source: Springer protocols feed by Pharmacology/Toxicology - May 1, 2014 Category: Drugs & Pharmacology Source Type: news
Automated Cell-Free Protein Production Methods for Structural Studies
In contrast to cell-based protein expression, cell-free production is highly consistent, scalable, and amenable to automation. Robots can handle many samples and perform repetitive procedures that are otherwise prone to human error. Here is described commercially available robotics for a wheat germ cell-free system with emphasis on practical applications for structural and functional studies. In addition, described is a cell-free method for preparing protein complexes. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Eukaryotic Expression Systems for Structural Studies
This protocol describes protein production in mammalian cells by transient transfection. It assumes the expression construct contains either a 6-HIS or Fc fusion tag to allow recovery of the protein by affinity chromatography. The method is one of the simplest available for protein expression in eukaryotic cells, requires little specialized equipment, and has a reasonably high rate of success. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Protein Production for Structural Genomics Using E. coli Expression
The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large qua...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Expression and Solubility Testing in a High-Throughput Environment
The expression and screening of the solubility of recombinant proteins is an important step in the high-throughput (HT) production of target proteins. For many applications, E. coli remains the most widely used expression system due to the relative ease of adapting it to HT pipelines. Herein is described a platform using a 96-well format for efficient expression and solubility screening of target proteins. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
High-Throughput Cloning for Biophysical Applications
Efficient high-throughput gene cloning represents a critical first step for conducting functional and structural proteomics in the post-genomic era. The ligation-independent cloning (LIC) method has been almost universally adopted by large structural biology centers as a component of high-throughput structure determination pipelines. The LIC platform is easy to use, of low cost, and rapid, and importantly, it is easily adapted to 96- or 384-well format, thereby facilitating automation. Procedures are described for 96-well format cloning using the LIC technology. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Selecting Targets from Eukaryotic Parasites for Structural Genomics and Drug Discovery
The selection of targets is the first step for any structural genomics project. The application of structural genomics approaches to drug discovery also starts with the selection of targets. Here, three protocols are described that were developed to select targets from eukaryotic pathogens. These protocols could also be applied to other drug discovery projects. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Target Selection for Structural Genomics of Infectious Diseases
This chapter describes the protocols used to identify, filter, and annotate potential protein targets from an organism associated with infectious diseases. Protocols often combine computational approaches for mining information in public databases or for checking whether the protein has already been targeted for structure determination, with manual strategies that examine the literature for information on the biological role of the protein or the experimental strategies that explore the effects of knocking out the protein. Publicly available computational tools have been cited as much as possible. Where these do not exist,...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Structural Genomics of Human Proteins
Structural genomics efforts focused on the human proteome have had three aims: to understand the structural and functional variations within protein families; to understand the structural basis of disease and genetic variation; and to determine the structures of human integral membrane proteins. The overarching theme is to advance the understanding of human health and to provide a structural platform to aid in the development of therapeutics. A decade or more of work in this field has identified optimal experimental strategies that can be used to expedite expression and crystallization of human proteins—and we provid...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Case Study—Structural Genomics and Human Protein Kinases
A brief summary is made of some of the methods in structural genomics and drug discovery as they apply to protein kinases. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Screening Ligands by X-ray Crystallography
X-ray crystallography is an invaluable technique in structure-based drug discovery, including fragment-based drug discovery, because it is the only technique that can provide a complete three dimensional readout of the interaction between the small molecule and its macromolecular target. X-ray diffraction (XRD) techniques can be employed as the sole method for conducting a screen of a fragment library, or it can be employed as the final technique in a screening campaign to confirm putative “hit” compounds identified by a variety of biochemical and/or biophysical screening techniques. Both approaches require an ...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Ligand Screening Using NMR
NMR has proven to be an invaluable technique for identifying and characterizing ligand interactions with biomolecules. NMR-based detection of ligand binding to protein targets is described. Specifically, the use of the WaterLOGSY NMR experiment to screen mixtures of compounds from a fragment library for binding to influenza H5 hemagglutinin is detailed. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Ligand Screening Using Enzymatic Assays
An important aspect of enzymatic assays is that the effect of a ligand on enzyme activity is readily apparent and quantifiable. Enzyme-based assays are, therefore, highly amenable to high-throughput ligand screening, which profiles the effect of a panel of small molecules on a designated target. In order for enzyme assays to provide useful screening data, the kinetics, assay components, readout signal, and overall stability of the assay are optimized and adapted to the equipment prior to the screen. For the screen itself, careful consideration is given to the number of replicates, the plate layout, the compound concentrati...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Ligand Screening Using Fluorescence Thermal Shift Analysis (FTS)
The fluorescence thermal shift (FTS) method is a biophysical technique that can improve productivity in a structural genomics pipeline and provide a fast and easy platform for identifying ligands in protein function or drug discovery screening. The technique has gained widespread popularity in recent years due to its broad-scale applicability, throughput, and functional relevance. FTS is based on the principle that a protein unfolds at a critical temperature that depends upon its intrinsic stability. A probe that will fluoresce when bound to hydrophobic surfaces is used to monitor protein unfolding as temperature is increa...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Data Management in the Modern Structural Biology and Biomedical Research Environment
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data. The traditional method of data reduction is very simple—results are summarized in peer-reviewed publications, which are hopefully published in high-impact journals. By their nature, publications include only the most important results derived from experiments that may have been performed over the course of many years. The main content of the published paper is a concise compilation of these data, an interpretation of the experimental results, and a comparison of these results with those obtained by other scientists. ...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
