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Results for “High Throughput Screening”

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At least 613 records · Page 34Linked to original sources
PubMed2006

Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery.

Insect cell-based expression systems are prominent amongst current expression platforms for their ability to express virtually all types of heterologous recombinant proteins. Stably transformed insect cell lines represent an attractive alternative to the baculovirus expression system, particularly for the production of secreted and membrane-anchored proteins. For this reason, transformed insect cell systems are receiving increased attention from the research community and the biotechnology industry. In this article, we review recent developments in the field of insect cell-based expression from two main perspectives, the production of secreted and membrane-anchored proteins and the establishment of novel methodological tools for the identification of bioactive compounds that can be used as research reagents and leads for new pharmaceuticals and insecticides.

Animals↗
PubMed2005

In vitro SCFbeta-Trcp1-mediated IkappaBalpha ubiquitination assay for high-throughput screen.

An increasing body of evidence indicates that constitutive activation of NF-kappaB contributes to tumorigenesis and inflammation. Ubiquitination and degradation of IkappaB plays an essential role in NF-kappaB activation. Here we describe an in vitro IkappaBalpha ubiquitination assay system in which purified E1, E2, SCF(beta-Trcp1) E3, IkappaBalpha, IKK2, and Ub were used to generate ubiquitinated IkappaBalpha. The ubiquitination of IkappaBalpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against IkappaBalpha was used to capture IkappaBalpha, and the biotinylated ubiquitin attached to IkappaBalpha was detected with europium (Eu)-labeled streptavidin. This assay can be used to discover inhibitors of IkappaBalpha ubiquitination. Such inhibitors would block NF-kappaB activation by stabilizing IkappaB levels in cells and thus provide a new therapeutic approach to NF-kappaB-related human diseases.

Blotting, Western↗
PubMed2006

Cell imaging assays for G protein-coupled receptor internalization: application to high-throughput screening.

There are a number of assays currently available to study G protein-coupled receptors (GPCRs), including ligand binding and functional assays. The latter category, albeit more complex, offers some obvious advantages over traditional ligand-binding assays. Functional cell-based assays typically include second messenger and reporter gene assays, which depend directly or indirectly on the cellular signaling cascade initiated upon receptor activation, respectively. More recently, cell imaging assays monitoring receptor trafficking are becoming increasingly popular. These assays, described in greater detail in this chapter, are independent of receptor signaling and are thus ideally suited for orphan receptors. In addition, these assays provide a valuable measure of receptor desensitization, an important feature for the use of GPCR agonists as potential therapeutic agents. The most popular GPCR imaging assays are based on the principles of receptor desensitization and internalization monitored directly or indirectly by green fluorescent protein.

Animals↗
PubMed2006

High-throughput screening for modulators of stem cell differentiation.

Realizing the potential of stem cell biology requires the modulation of self-renewal and differentiation, both of which are incompletely understood. This chapter describes methods for the design, development, and implementation of cell-based screens of small molecules, genes and expressed proteins for modulation of stem and progenitor cell fate. These include the engineering of embryonic and other stem cells with gene promoter-reporter protein constructs and their application in automated screening. We discuss considerations of promoter reporter selection, assay development and implementation, and image acquisition, analysis, and data handling. Such black-box screens are useful for the identification of probes of developmental processes and should provide tools that will identify druggable targets for biochemical assays.

Animals↗
PubMedDate not supplied

High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay.

Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2-3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring approximately 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 microg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 microg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 microg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring <1 mg test material, giving a substantial increase in productivity. Any material selected for further development and inclusion in a formulation may require further confirmatory testing, e.g. using a human skin model assay for phototoxicity.

Animal Testing Alternatives↗
PubMed2000

High-throughput screening of enzyme libraries.

Directed evolution is becoming a widely used technique for modifying or enhancing protein performance. Ultimately, the success of directed protein evolution experiments hinges on the efficiency of the methods used to screen libraries for mutants with properties of interest. Although there is still a paucity of general methods for enzyme library screening, in recent years a number of promising strategies have emerged and are increasingly being used to explore challenging issues in protein engineering.

Directed Molecular Evolution↗
PubMed1998

Intracellular detection assays for high-throughput screening.

New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Most of these methods employ either fluorescent or luminescent read-outs and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Furthermore, genetically encoded probes offer the possibility of custom-engineered biosensors for intracellular biochemistry, specifically localized targets, and protein-protein interactions.

Animals↗
PubMed2003

A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases.

The water soluble fluorescein-based ligand 1 forms a non-fluorescent complex with Cu(2+). This complex serves as a fluorescent sensor for amino acids in the 10(-3) M concentration range. Since the signal response is very fast, the sensor can be used to detect the hydrolytic activity of various proteases (trypsin, chymotrypsin, subtilisin) on bovine serum albumin as a whole protein substrate, and more generally to follow reactions releasing or removing free amino acids, in real time.

Amino Acids↗
PubMed2003

A low background high-throughput screening (HTS) fluorescence assay for lipases and esterases using acyloxymethylethers of umbelliferone.

Esters and acyloxymethyl ethers of umbelliferone were evaluated as fluorogenic substrates for lipases and esterases with respect to stability and resistance to non-specific hydrolysis. Isobutyryloxymethyl ether 1c and pivaloxymethyl ether 1d were found to be optimal substrates for enzyme assays, particularly with respect to HTS applications.

Drug Evaluation, Preclinical↗
PubMed2000

High-throughput screening brings in the crops.

Screening is the acknowledgement of ignorance. Such a provocative statement might not amuse our community and, in fact, an incredible quantity of knowledge does go into each screen. However, from a structural point of view, screening is where serendipity meets prediction.

Journal Article↗
PubMed2003

Confocal optics microscopy for biochemical and cellular high-throughput screening.

In recent years, both academia and pharmaceutical industry have produced significant advances in confocal detection and spectroscopy by laser-induced fluorescence. Confocal fluorescence studies provide information on identity, size, diffusion coefficient and concentration of the fluorescently labeled entity. This enables the establishment of sophisticated biochemical drug screening assays using the multitude of fluorescence parameters that can be observed (e.g. molecular brightness, fluorescence lifetime, anisotropy, resonance energy transfer). In cellular screening assays, confocality introduces spatial resolution in the vertical direction and reduces background fluorescence from outside the focal plane. Confocal HTS systems focusing on femtoliter-sized observation volumes allow for assay volumes far beyond current limits.

Microscopy, Confocal↗