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Past Issue in 2013
Volume: 3, Issue: 11
Developmental Biology
Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos
Fluorescent conjugated Phalloidin is a stain that allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest. Here is a protocol for Phalloidin and antibody staining of zebrafish embryos 5 days old and younger.
Motility Assay for Zebrafish Embryos
Analyzing the swimming ability of 2 days post fertilization zebrafish embryos can be a useful technique to study neuromuscular function. Here is a protocol for determining the time it takes for zebrafish embryos to swim a predetermined distance.
Immunology
Generation of Polyclonal Specific Antibodies
Generation of antibodies specific for a protein of interest is a common method in many disciplines. This protocol details the steps in production of a polyclonal antibody in rabbits using a bacterially expressed fusion protein as an antigen. The protocol is generated based on data presented in Wirschell et al.(2013).
Microbiology
Detection of Membrane Potential in Mycobacterium tuberculosis
DiOC2 (Novo et al., 2000) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts towards red emission as the dye molecules self associate at the higher cytosolic concentrations caused by larger membrane potentials. Proton ionophores such as CCCP destroy membrane potential by eliminating the proton gradient. The magnitude of membrane potentials varies with different bacterial species. For many gram-positive species, including Staphylococcus aureus and Micrococcus luteus, the red:green ratio tends to vary with the intensity of the proton gradient while in many gram-negative bacteria such as Escherichia coli and Salmonella choleraesuis, the response of the dye does not appear to be proportional to proton gradient intensity. Mycobacterium tuberculosis itself is a difficult organism to work with because of its rigid cell wall.
Quantification of Retro- and Lentiviral Reverse Transcriptase Activity by Real-time PCR
Quantification of retroviral reverse transcriptase activity in retrovirus containing supernatant by quantitative reverse transcription PCR as a method for titration of HIV, lenti- and retroviral vectors is described here.. The procedure was optimized for use with LightCycler 480 (Roche, Vilvoorde, Belgium) and ABI 7300 real-time PCR system (reagents and procedures that are system specific will be marked accordingly in the protocol).
Neuroscience
Electroolfactogram (EOG) Recording in the Mouse Main Olfactory Epithelium
Olfactory sensory neurons in the main olfactory epithelium (MOE) are responsible for detecting odorants and EOG recording is a reliable approach to analyze the peripheral olfactory function. However, recently we revealed that rodent MOE can also detect the air pressure caused by airflow. The sensation of airflow pressure and odorants may function in synergy to facilitate odorant perception during sniffing. We have reported that the pressure-sensitive response in the MOE can also be assayed by EOG recording. Here we describe procedures for pressure-sensitive as well as odorant-stimulated EOG measurement in the mouse MOE. The major difference between the pressure-sensitive EOG response and the odorant-stimulated response was whether to use pure air puff or use an odorized air puff.
Plant Science
2D Diagonal Redox SDS-PAGE of Proteins
2D diagonal redox SDS-PAGE of proteins is used to detect intramolecular or intermolecular disulfide bridges using Chlamydomonas in this example (Stroeher and Dietz, 2008; Schwarz et al., 2012). Both dimensions consist of a conventional SDS-PAGE, except that the sample buffer for the first dimension lacks a reducing agent. Intermolecular disulfide bridges increase the apparent molecular weight of a protein in the first dimension, whereas intramolecular bridges decrease the apparent weight of the protein.
Wax Analysis of Stem and Rosette Leaves in Arabidopsis thaliana
The primary aerial surfaces of all land plants are coated by a lipidic cuticle, which restricts non-stomatal water loss and protects the plant from pathogens, herbivores, and ultraviolet radiation. The cuticle is made up of two components: cutin, a polymer of hydroxy- and epoxy- long-chain fatty acid derivatives and glycerol, and cuticular waxes, which are derivatives of very-long-chain fatty acids. While chemical analysis of cutin can be a lengthy and technically challenging task, analysis of cuticular waxes is relatively simple, and can be routinely used to characterize different plant species, adaptations of a given species to environmental conditions, or mutant phenotypes. Here, we present a protocol tailored for the analysis of cuticular waxes on the surface of the model organism Arabidopsis thaliana. Because cuticular waxes are found on the outermost surface of the plant, the wax extraction process is very simple, and sample processing can be completed in less than one day. Chemical analysis involves quantitation of wax monomers by gas chromatography coupled with flame ionization detection (GC/FID), and identification of wax monomers by either mass spectrometry or comparison of retention times of individual wax components to those of known standards.
35S pulse Labelling of Chlamydomonas Chloroplast Proteins
35S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of radioactive sulfur. This example uses Chlamydomonas cells and H235SO4 (Schwarz et al., 2012).
Systems Biology
Detection of DNA Methylation Changes Surrounding Transposable Elements
Transposable elements (TEs) are a major component of all genomes, thus the epigenetic mechanisms controlling their activity is an important field of study. Cytosine methylation is one of the factors regulating the transcription and transposition of TEs, alongside Histone modifications and small RNAs. Adapter PCR-based methods [such as Amplified Fragment Length Polymorphism (AFLP)] have been successfully used as high-throughput methods to genotype un-sequenced genomes. Here we use methylation-sensitive restriction enzymes, in combination with PCR on adaptor-ligated restriction fragments, to evaluate epigenetic changes in TEs between genomic DNA samples.
High Resolution Detection of Genetic Changes Associated with Transposons
Transposable elements (TEs) are repetitive sequences, capable of inducing genetic mutations through their transpositional activity, or by non-homologous or illegitimate recombination. Because of their similarity and often high copy numbers, examining the effects of mutations caused by TEs in different samples (tissues, individuals, species, etc.) can be difficult. Thus, high throughput methods have been developed for genotyping TEs in un-sequenced genomes. A common method is termed Transposon Display (or transposon SSAP), which utilizes restriction enzymes and PCR amplification to produce chimeric DNA molecules that include genomic and TE DNA. The advent of second generation sequencing technologies, such as 454-pyrosequencing, have dramatically improved the resolution of this assay, allowing the simultaneous sequencing of all PCR products, representing all amplified TE sites in a specific genome.