detected Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Annexin V, belonging to a recently discovered family of proteins, the annexins, with anticoagulant properties has proven to be a useful tool in detecting apoptotic cells since it preferentially binds to negatively charged phospholipids like PS in the presence of Ca2+ and shows minimal binding to phosphatidylcholine and sphingomyeline. Changes in PS asymmetry, which is analyzed by measuring Annexin V binding to the cell membrane, were detected before morphological changes associated with... - [Read Annexin V Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other manufacturers may work equally well but have not been tested. Includes: Labeling of BAC clones; Ethanol precipitation; Hybridization; Post-hybridisation treatment / detection. - [Read BAC-FISH Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

This bioassay utilizes cultured Hepa-lclc7 (Hepa-1) mouse hepatoma cells to assess the CYPlA1-inducing potency or cytotoxicity of pure test chemicals or environmental samples.  In the Hepa-l induction test , the CYPlA1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities. - [Read CYP1A1-Inducing Potency and Cytotoxicity Test in the HEPA-1 Mouse Hepatoma Cell Line]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Changes in the balance of cytoskeletal proteins after exposure to test compounds can be detected by indirect immunofluorescence microscopy and quantitative biochemical methods. - [Read Cytoskeletal Alterations as a Parameter for Assessment of Toxicity]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol uses Rnase protection to detect short interfering RNAs (siRNAs) in RNA preparations from Caenorhabditis elegans.  SiRNAs can also be detected by northern blot.  However, the Rnase protection assay seems to be more sensitive. - [Read Detection of siRNA in C. elegans Using Rnase Protection Protocol]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

DNA laddering can be detected from samples with only 8% apoptotic cells. Alternatively, the cells can be stained with DAPI and analysed by flow cytometry. CellDeath.de - [Read DNA laddering assay for treated cells]

Category: Antibody Protocols: Epitope Mapping Antibody Protocols

A set of overlapping synthetic peptides is synthesized, each corresponding to a small segment of the linear sequence of a protein antigen and arrayed on a solid phase. The panel of solid-phase peptides is then probed with a test antibody, and bound antibody is detected using an enzyme-labeled secondary antibody. This method is very rapid and can be extraordinarily successful. - [Read Epitope Mapping Using Synthetic Biotin-Labeled Peptides Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. - [Read Flow Cytometry of Apoptosis Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes a method for establishing short-term explant cultures of oesophageal mucosa. Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. - [Read Human Oesophageal Culture Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Protocol describes a method for establishing short-term explant cultures of oesophageal mucosa.  Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. - [Read Human Oesophageal Culture Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents.  Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags.  Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. - [Read Immunoblotting: Antigen Detection Using Chemiluminescence Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents.  Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. - [Read Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to fluorophore. - [Read Immunofluorescence Staining Protocol]

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Immunoprecipitation Troubleshooting Guide. Stressgen. Problem: Non-specific background, Specific Background, Specific antigen not detected. With Solutions to common IP problems. - [Read Immunoprecipitation Troubleshooting Guide PDF]

Category: Molecular Biology Protocols: Carbohydrate Protocols

Immunostaining thin layer chromatograms TLC is a very sensitive detection technique of functionally active carbohydrate ligands of protein receptors. Carbohydrate structures are detected in glycolipids from complex mixtures of molecules extracted from the relevant target tissue. Proteins analyzed can be antibodies, chimeric Ig proteins, selectins, lectins, toxins, and other carbohydrate binding proteins. John L. Magnani~GlycoTech Corporation, Rockville, Maryland - [Read Immunostaining Thin Layer Chromatograms Of Glycolipids]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

During incubation period and the course of disease, SARS virus replicates and releases from cells. The level of virus varies greatly from sample to sample. At early stage of incubation period or during the late convalescent phase, the concentration of virus is very low in all kinds of samples. Virus is also detected at extremely low concentrations in plasma during... - [Read Kit for Rapid Enrichment of SARS Virus]

Category: Antibody Protocols: Antibody Labeling Protocols

This protocol describes the covalent coupling of antibodies to biotin. Biotin groups bind with extremely high affinity to streptavidin and avidin, both of which are available commercially coupled with enzymes, fluorescent dyes, or iodine. A biotinylated primary antibody, therefore, can be detected with any of a wide variety of different labels. The biotinylation reaction is simple and mild, and rarely inactivates the antibody. - [Read Labeling Antibodies with Biotin Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

This protocol describes a simple chemical oxidation method for labeling antibodies with iodine. Iodide-125 (supplied as NaI) is oxidized to form iodine-125 (I2), which attacks tyrosyl and histidyl side chains. The iodinated antibodies are easily detected and quantitated using gamma counters or film. They are used primarily in immunoassays, but other techniques can be adapted conveniently to the iodine detection method. - [Read Labeling Antibodies with Iodine Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

In this test rabbit articular chondrocytes are cultured in the presence of test compound, the toxicity of which is then determined by its effect on the production of proteoglycan by the cells, as detected by the dye Alcian Blue. - [Read Rabbit Articular Chondrocyte Functional Toxicity Test]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs. - [Read Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

In an attempt to accurately measure DNA content with simultaneous preservation of cell surface markers, we have utilized gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association. For some cell types, the presence of apoptotic cells based on reduced DNA content can also be detected. One such technique employs the addition of ethanol to cells previously resuspended in high concentrations of fetal bovine serum... - [Read Simultaneous Analysis of DNA Content and Surface Immunophenotype Protocol]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

The target sequence is simultaneously labeled and amplified, then heat-denatured and resolved by non-denaturing polyacrylamide gel electrophoresis.  Differences in sequence alter the conformation of the DNA and hence its electrophoretic mobility and, because of the high resolution of polyacrylamide gels, most conformational changes caused by subtle changes in sequence can be detected. - [Read Single-Strand Conformation Polymorphism Analysis Protocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]