developed Protocols

Category: DNA Protocols: EMSA DNA-Protein Protocols

A non-radioactive electrophoretic mobility shift assay based on the digoxigenin detection system had been developed and applied to the analysis of the interaction between the heat shock transcription factor and the heat shock element of N. crassa. (Bioch - [Read A non-radioactive electrophoretic mobility shift assay for the detection of heat shock element (HSE)]

Category: Fungi Protocols: Fungi Genetics Protocols

Protocol describes a safe and convenient method of extracting DNA from Coccidioides immitis fungi in which the culture is killed by steaming, allowing removal from the containment facilities, as soon as possible. The method was first developed with the non-pathogen Neurospora crassa, has worked well for both C. immitis and H. capsulatum, and should be useful for extracting DNA from any pathogenic fungus. - [Read A Safe Method of Extracting DNA from Coccidioides immitis Protocol]

Category: PCR Protocols: AFLP PCR

AFLP® technology was developed initially by Keygene in the early 1990’s. AFLP has become a widespread genetic fingerprinting technology. AFLP video and diagrams. Keygene - [Read AFLP - Genetic Fingerprinting]

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: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Protocol for chromosomal DNA preparation. Protocol was developed for cultured cells but should be appropriate for dissociated tissues as well. - [Read Chromosomal DNA Preparation Protocol]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Shaw et al. Fungal Genetics and Biology 2001. first report of a plant pathogenic fungus for which aequorin-based Ca21 measurement protocols have been developed. - [Read Expression of Recombinant Aequorin as an Intracellular Calcium Reporter PDF]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. As the cells age, so their density tends to increase; reticulocytes therefore tend to have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite varied, from quite broad ones. - [Read Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol]

Category: Lipid Protocols

New screening efforts and chemical modifications of existing compounds have been attempted to identify more selective and potent inhibitors. To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel-elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Developed PCR-based single molecule haplotyping methods that enable both surveys for novel inversion variants, and population-scale genotyping of known inversions - [Read Haplotype-Fusion PCR Protocol]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol describes the direct detection of RNA on DNA microarrays using Hybrid Capture (HC) technology and the HC ExpressArray Kit developed by Diagene.  The kit uses a proprietary antibody that binds specifically to RNA:DNA hybrids and a second, fluorescently labeled, antibody that detects the primary antibody.  Total RNA is applied directly to a glass-spotted DNA microarray, and stable RNA:DNA hybrids are visualized via a Cy3-labeled secondary antibody. - [Read Hybridization and Detection Using the HC ExpressArray Kit Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

There are several strategies to visualize the antibody. For transmitted light microscopy, color development substrates for enzymes are often used. The antibody can be directly labeled with the enzyme. However, such a covalent link between an antibody and an enzyme might result in a loss of both enzyme and antibody activity. For these reasons several multistep staining procedures have been developed, where intermediate link antibodies are used. In this protocol use the Vectastain ABC-kit. - [Read Immunocytochemistry in Free-Floating Sections Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

To accurately predict the activity of a transgene it is critical to understand its location and dynamics in the 3-D interphase nucleus. Developed in situ methods to visualize transgenes (including single copy genes) & their transcripts during interphase from different tissues & plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis and extend characterization to the interphase nucleus - [Read In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei]

Category: Bacterial Protocols

Protocol was developed to isolate Wolbachia from adult Drosophila, but it can be adapted for other insects. In some insects leg removal prior to isolation facilitates hemolymph extrusion. - [Read Isolation of Live Bacteria from Adult Insects Protocol]

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

This simple cell culture-based cytotoxicity test (in which cell viability is determined by uptake of the dyes ethidium bromide and fluorescein acetate) has been developed as a general test for acute toxicity. - [Read LS-L929 Cytotoxcitiy Test]

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

Measurement of Apoptosis and Other Forms of Cell Death. Jagan Muppidi, Melissa Porter, and Richard M. Siegel. As programmed cell death (PCD) or apoptosis has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. - [Read Measurement of Apoptosis and Other Forms of Cell Death]

Category: Immunology

A method for ameliorating autoimmune disease by passive transfer of IVIg-primed leukocytes. Developed a method for ‘priming’ specific leukocyte populations with IVIg to study their potential role in ameliorating ITP upon passive transfer, which we describe herein. Using this technique, found that splenic CD11c+ dendritic cells reacted with IVIg, or several IVIg mimetic regimes, were primed to ameliorate ITP upon transfer to thrombocytopenic mice. - [Read Method for Ameliorating Autoimmune Disease by Passive Transfer of IVIg-Primed Leukocytes]

Category: DNA Microarray Protocols: DNA Methylation Microarray CpG

Kimura et al., NAR 2005. "easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylatio - [Read Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorec]

Category: Cytogenetics Protocols

Protocol describes a recently developed method — methylation-specific digital karyotyping (MSDK) — that enables comprehensive and unbiased genome-wide DNA methylation analysis. Using a combination of a methylation-sensitive mapping enzyme (for example, AscI) and a fragmenting enzyme (for example, NlaIII), short sequence tags can be obtained and uniquely mapped to genome location. - [Read Methylation-Specific Digital Karyotyping Protocol]

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

Human tissues are comprised of multiple interacting cell populations in a complex three dimensional arrangement with each cellular phenotype determined by a unique profile of mRNA and protein expression. Before microdissection techniques were developed, the only analysis tools for phenotypic studies were primarily immunohistochemistry and in-situ hybridization. While useful, these tools are limited to single gene analysis and, in general, do not allow qualitative studies. - [Read Microdissection Overview]

Category: Protein Protocols: Protein Precipitation Procols

Protein Precipitation. Developed by: Eric Burtson. American Association of Immunologists 1995. In this lab you will separate a solution of proteins using protein precipitation. Since you will be testing the same protein mix that you used in the Gel Filtration lesson, you will pass the protein through a gel filtration column to identify which protein(s) precipitates. - [Read Protein Precipitation]

Category: Signalling Signal Transduction Protocols

Detection of phosphorylated tyrosine residues can be performed using anti-P-TYR Ab and Western Analysis.Includes 2nd method,which uses phosphotyrosine in conjunction with anti-P-TYR Ab to "unlabel" potential proteins.By comparing Westerns developed with the 1st method(reveals phosphorylated protein) and the 2nd method(reveals non-specific labeling), a more accurate picture of those proteins phosphorylated on tyrosine can be seen. Includes: Protein Preparation, Electrophoresis and Transfer. - [Read Protocol for Antiphosphotyrosine Western Blot Analysis]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

Category: RNA Protocols: cDNA Libraries Library

Munroe et al PNAS 1995 "..developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. - [Read Systematic screening of an arrayed cDNA library by PCR PDF]

Category: Neuroscience Protocols: Patch Clamping Protocols

The cell-attached capacitance recording technique is a powerful technique that has been successfully used to resolve single vesicle fusion and fusion pore conductance.  This technique, however, has not been applied to synapses because of the difficulty in accessing release sites.  Here, we developed a technique to expose release sites in a large nerve terminal, the calyx of Held, which contains clear-core glutamatergic vesicles. - [Read The Cell Attached Capacitance Recording Technique]