detect Protocols

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

Describes an experimental cross in mice that can be used to define and map induced germ-line mutations that map to a single chromosome. The cross is a modification and extension of a conventional three-generation recessive mutagenesis screen. Includes: The Mutagenesis Breeding Plan; Consomic Strains; Generating Mutations; Generating and Genotyping G2 Females; Genotyping G3 Progeny; Phenotyping G4 Progeny; etc.. - [Read A Targeted Screen to Detect Recessive Mutations that have Quantitative Effects Protocol]

Category: Cell Organelle Protocols: Mitochondria Protocols

The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. - [Read Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Protocol and methods on assays to detect apoptosis. - [Read Assays to Detect Apoptosis Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

This unit describes how to detect and recognize bacterial, fungal, or mycoplasma contamination and how to verify the presence of mycoplasma. Strategies are described for dealing with culture contamination, if necessary. - [Read Assessing and Controlling Microbial Contamination in Cell Cultures Protocol]

Category: ELISA Protocols: Sandwich ELISA Assay Protocols

Cytokine sandwich ELISA are sensitive enzyme immunoassays that can specifically detect and quantitate the concentration of soluble cytokine and chemokine proteins. BD Biosciences - [Read Cytokine ELISA Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Three Ambion kits were used to quantitate specific miRNAs and to detect differential miRNA expression in various mouse brain regions and cell types isolated by laser capture microdissection (LCM). These techniques can be applied to studying miRNA in other species, tissues, and cell types. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Category: RNAi Technology Protocols

Information on how detect and quantitate MicroRNA in laser capture microdissection samples. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR; Detection of miRNA in Microdissected Tissue from Mouse Brain by qRT-PCR; Differential Expression of MicroRNA in Whole Brain Tissue Compared to a More Homogeneous Population of Cells. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

The most convenient and commonly used method to visualize DNA in agarose gels is staining with the fluorescent dye ethidium bromide.  Ethidium bromide can be used to detect both singleand double-stranded nucleic acids (both DNA and RNA).  However, the affinity of the dye for single-stranded nucleic acid is relatively low and the fluorescent yield is comparatively poor. - [Read Detection of DNA in Agarose Gels Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of even-skipped transcripts in drosophila embryos with PCR/DIG-labeled DNA probes. This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. Includes: Probe labeling; Evaluation of labeling reaction; Preparation of embryos, hybridization and detection.
 - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes. Protocol was optimized from a protocol using 35S-labeled RNA probes. It allows to detect the expression of low abundant mRNAs in the cardiovascular system, e.g. of the proinflammatory cytokine GM-CSF in normal human coronary arteries, and of IL6 and gp130 in human failing hearts. The protocol can be combined with immunohistochemistry. - [Read Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol uses northern blot technology to detect short interfering RNAs (siRNAs) or micro-RNAs (miRNAs) in RNA preparations from Caenorhabditis elegans. - [Read Detection of si/miRNA in C. elegans by Northern Blot Protocol]

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: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes.  The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization.  This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. 
    - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG. - [Read Epitope Tagging of Recombinant Proteins Protocol]

Category: Developmental Biology: Embryology Protocols

The FAM caspase binding assay kits from ATCC Corporation can be used to determine amounts of active caspases in cells. The FAM-labeled caspase inhibitor can freely diffuse into the cell. Active caspase irreversibly binds the inhibitor. Upon washing the cells, the amount of fluorescence is proportional to the amount of active caspase in the cell. FAM-LETD-fmk (catalog no. 30-1306) is used to detect caspase 8 and FAM-LEHD-fmk (catalog no. 30-1308) is used for caspase 9. - [Read Fam Caspase 8 and 9 Binding Assay for Embryos Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

This method of first strand cDNA synthesis is more efficient than Ready-to-Go beads.  You can use as little as 100ng total RNA and still detect you gene expression in PCR. - [Read First strand cDNA synthesis with Super Script II Protocol]

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

This test is designed to detect irreversible toxic effects on both cell growth and survival, by the evaluation of colony-forming (CF) efficiency, in hepatoma cell lines derived from man, rat and mouse. - [Read Hepatoma Cell Cultures as In Vitro Models for Hepatotoxicity]

Category: Immunology: Immunofluorescence Protocols

This protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells. Western blot technology can be used as an alternative (see Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting). - [Read Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells.  Western blot technology can be used as an alternative. - [Read Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol]

Category: Signalling Signal Transduction Protocols: MAPK Protocols

Protocol describes an in vitro assay to detect MAP kinase activity using PC12 cells. - [Read In Vitro MAP Kinase Assay]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Leukostat Staining of Cytospin Preparations to Detect Apoptosis. Shailaja Kasibhatla et al. Leukostat staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a light microscope and counted to quantify apoptosis. This protocol can be used both for cells that grow in suspension and for adherent cells. - [Read Leukostat Staining of Cytospin Preparations to Detect Apoptosis]

Category: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

This method is used to detect genomic DNA deletions in tumor cells. - [Read Loss of Heterozygosity Protocol]

Category: Protein Microarray Protocols

Lysate Microarrays. High throughput methods that detect abundance and post-translational modification. MacBeath Lab - [Read Lysate Microarrays - Information]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

One of the most important, but frequently overlooked, cell culture procedures is testing cultures for microbial contamination, especially mycoplasma. It is critical for every cell culture laboratory to only use cell lines that have been carefully screened for mycoplasma. Fortunately, there is a simple fluorochrome DNA staining test that can detect both mycoplasma and virtually any other prokaryote contaminants. - [Read Mycoplasma Detection Using DNA Staining Protocol]