The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Lewis A Chodosh - One of the best experts on this subject based on the ideXlab platform.
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mobility shift dna Binding Assay using gel electrophoresis
Current protocols in pharmacology, 2001Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
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mobility shift dna Binding Assay using gel electrophoresis
Current protocols in molecular biology, 1996Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:The DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
Stephen Buratowski - One of the best experts on this subject based on the ideXlab platform.
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mobility shift dna Binding Assay using gel electrophoresis
Current protocols in pharmacology, 2001Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
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mobility shift dna Binding Assay using gel electrophoresis
Current protocols in molecular biology, 1996Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:The DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
Mareli Kelderman - One of the best experts on this subject based on the ideXlab platform.
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Performance and utility of a cost-effective collagen-Binding Assay for the laboratory diagnosis of Von Willebrand disease.
Clinical chemistry and laboratory medicine, 2007Co-Authors: Muriel Meiring, Philip N. Badenhorst, Mareli KeldermanAbstract:Background: The collagen-Binding Assay, a functional Assay of Von Willebrand factor (VWF), discriminates the subtypes of Von Willebrand disease (VWD). Commercial collagen Binding Assays have the advantage of immediate use for fast results, but are expensive. Methods: In this study we evaluated an in-house collagen-Binding Assay using type III collagen. We included it in the diagnostic work-up of 44 patients with VWD and 40 normal subjects. Other Assays included VWF antigen, ristocetin cofactor activity, ristocetin-induced platelet agglutination and VWF multimeric analysis. Results: The cost of this collagen-Binding Assay is 10-fold lower than that of commercial kits. The intra- and inter-Assay coefficients of variation were
Jordi Molgó - One of the best experts on this subject based on the ideXlab platform.
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Coupling the torpedo microplate-receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins.
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor Binding Assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor Binding Assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.
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A non-radioactive ligand-Binding Assay for detection of cyanobacterial anatoxins using Torpedo electrocyte membranes.
Toxicon, 2008Co-Authors: Rómulo Aráoz, Jordi Molgó, Michael Herdman, Rosmarie Rippka, Aurélie Ledreux, Jean-pierre Changeux, Nicole Tandeau De Marsac, Hoàng-oanh NghiêmAbstract:Anatoxin-a (ANTX) and homoanatoxin-a (HANTX), neurotoxins exclusively produced by cyanobacteria (LD(50) 200-250 microg kg(-1), i.p. mouse), are agonists of the nicotinic acetylcholine receptors (nAChRs) to which they tightly bind. We have exploited the high affinity of these neurotoxins for the nicotinic receptors to develop a non-radioactive ligand-Binding Assay using Torpedo electrocyte membranes and biotinylated alpha-bungarotoxin (Biotin-BgTx) as tracer for detection of this class of toxins. The affinity of the Torpedo nAChRs for Biotin-BgTx was determined by chemiluminescence (K(d)=1.2 x 10(-8)M Biotin-BgTx) or color development (K(d)=3.5 x 10(-8)M Biotin-BgTx). Binding of ANTX or HANTX to the nAChRs competitively inhibits the Binding of Biotin-BgTx to the receptors in a concentration-dependent manner (chemiluminescence: IC(50): 6.2 x 10(-8)M ANTX; color development: IC(50): 1.7 x 10(-8)M ANTX). The proposed method was validated by HPLC/MS with detection in the single ion recording mode. The non-radioactive ligand receptor-Binding Assay was successfully applied to the analysis of extracts prepared from cyanobacteria in culture and from natural habitats, as well as from aqueous samples. This method is suitable for ANTX and HANTX early survey of environmental samples since it requires minimal manipulations, is highly sensitive and gives consistent signal-to-noise ratios.
Johannes-peter Stasch - One of the best experts on this subject based on the ideXlab platform.
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Receptor Binding Assay for NO-independent activators of soluble guanylate cyclase.
Methods in molecular biology (Clifton N.J.), 2013Co-Authors: Peter Schmidt, Johannes-peter StaschAbstract:The characterization of the interaction between a ligand and its receptor is crucial for a broad variety of applications in academia as well as in the pharmaceutical industry. Although various sophisticated high-throughput technologies have been established to investigate the Binding of ligands to their receptors, classical filtration-based receptor Binding Assays still have some advantages when smaller number of samples need to be tested. Here we describe a technically easy, cheap, and reliable receptor Binding Assay that was successfully applied to determine the Binding constant of the NO-independent activator of soluble guanylate cyclase, cinaciguat, and the impact of other small molecules on its interaction with the enzyme.