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43 Current white paper about the topic fluorescence

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Predictor(TM) hERG Fluorescence Polarization Assay Kit performed on the PHERAstar/PHERAstar Plus

28-08-2008

The potential for cardiotoxic side-effects continues to challenge the development of small molecule based therapies. These intolerable side-effects are often precipitated by drug-induced long QT syndrome (LQT), which is often linked to blocking the human ether-a-go-go related gene (hERG) potassium channel. Although patch-clamp electrophysiology remains the gold standard for determining the interaction of compounds with the function of the hERG channel, radioligand displacement assays have proven to be a cost-effective initial alternative assay for hERG channel liability at early stages of compound development....

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Transfection of Human Tooth-Specific Lineage Cells with FuGENE® HD Transfection Reagent

22-08-2008

Similar to other epithelial appendages, teeth form through reciprocal signaling interactions between the ectodermally derived dental epithelium and the mesenchyme. These signaling interactions program enamel matrix-secreting ameloblasts and dentin-forming odontoblasts. To study the signaling network involved in odontogenesis, we co-cultured primary human fetal dental epithelial cells and dental pulp cells to reconstitute the reciprocal signaling....

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Interference and HAMA problems

Recognizing and avoiding of false results

16-06-2008

Immunoassays are bioanalytical methods based on antibodies. These include ELISA, EIA, RIA, Western blotting, protein arrays, lateral flow assays (immuno-chromatography), immunohistochemistry or immuno-PCR. Antibodies detect the substances (analytes) in immunoassays. This detection is always highly specific and only the analytes are detected and bound. This is the theory! Practically any antibody not only binds its analyte. It also binds several other substances, but with very different affinities....

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Transcriptor High Fidelity cDNA Synthesis Kit - Accuracy Meets RT-PCR

04-06-2008

Retroviral reverse transcriptases commonly used for cDNA synthesis exhibit a higher error rate than other DNA polymerases used in nucleic acid analysis techniques. This lack of accuracy leads to a significant number of base exchanges or frameshifts which are further propagated in a subsequent PCR reaction. With the introduction of a new high-accuracy reverse transcriptase, Roche Applied Science now offers a new tool to increase the fidelity of cDNA synthesis. The Transcriptor High Fidelity cDNA Synthesis Kit not only reduces the amount of errors in RT-PCRs; it also allows the reverse transcription of full-length cDNAs with high yield....

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Optimization of Transfection Conditions for Gene Expression, siRNA Knock-Down, and Live Cell Imaging Using FuGENE® HD Transfection Reagent

22-05-2008

The experimental method of introducing foreign mole­cules into cells is an important tool to facilitate studies in various biological fields. Transfection using lipophilic methods (liposomes or other cationic species) is a convenient means of introducing many different types of molecules (plasmid DNA, RNA, siRNA, and miRNA) into eukaryotic cells [1,2]. However, the optimal condition for incorporation depends on the type of transfection reagent and the cells being used, resulting in variable transfection efficiency and the extent of cell damage caused by transfection....

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High sensitive detection of follistatin by Imperacer®: an initial study on the way to analytical gene-doping tests

08-05-2008

For the understanding of interactions in the muscle-growth directing myostatin-pathway and therefore also for the establishment of high sensitive detection strategies for gene-doping, detailed studies of protein concentration levels in biological matrices and appropriate highly sensitive analytic techniques for quantitative monitoring of these biomarkers are required. In this work, the development of a high sensitive assay for follistatin based on the Imperacer® technology is described. The quantification limit in human serum was found at 60 pg/ml in only 6 µl sample volume. With this initial assay we demonstrate the first application in a set of analytical tests for a high sensitive myostatin pathway related profiling....

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Molecular Probes® NanoOrange® Assay Performed on BMG LABTECH FLUOstar OPTIMA Microplate Reader

08-05-2008

The field of proteomics has expanded dramatically in recent years with research on a whole host of organisms. Techniques such as two dimensional difference gel electrophoresis (2D DIGE) require the accurate quantitation of protein, as up to three labelled samples can be loaded on a single gel for comparison. Assay methods for determining protein quantitation include absorbance at 280 nm(1), the Bradford assay(2), Lowry assay(3), BCA method(4) and more sensitive assays such as Fluoroprofile® (Sigma-Aldrich) and NanoOrange® (Molecular Probes®)5()....

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Rapid High-Throughput Methylation Analysis Using the LightCycler® 480 System

07-05-2008

The LightCycler® 480 Instrument is a versatile platform for molecular studies. We assessed the performance of the instrument combined with the LightCycler® 480 High Resolution Melting Master mix for DNA methylation analysis using the methylation-sensitive high resolution melting (MS-HRM) methodology. This proved to be a robust platform for performing MS-HRM experiments, allowing rapid and high-throughput studies of DNA methylation....

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Dual Luciferase Reporter (DLR) Assay Certification on the Omega Series of Readers

29-02-2008

The Dual-Luciferase® Reporter Assay or DLR is widely used to study gene transcription and regulation. The DLR assay is a two step reaction that uses two luciferase enzymes, Firefly and Renilla. The Firefly reaction is initiated, followed by its quenching and the subsequent initiation of the Renilla reaction....

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Confocal Detection of NBT/BCIP In Situ Hybridization

Samples by Reflection Microscopy

18-02-2008

Confocal laser scanning reflection microscopy (CLSM) in combination with whole-mount in situ hybridization (WMISH) is a novel method to visualize gene expression patterns in three dimensions in whole embryos. The method uses conventional NBT/BCIP precipitation by alkaline phosphatase in WMISH samples and confocal detection by reflection microscopy. WMISH protocols can also be combined with fluorescent counterstainings such as DAPI, antibodies, GFP lines, and fluorescent in situ probes. Such double and triple labeling combined with confocal microscopy allows expression profiling at a cellular resolution in cell types or embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale, cellular resolution expression profiling in vertebrate and invertebrate model organisms....

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