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Past Issue in 2013
Volume: 3, Issue: 16
Biochemistry
Protein Flotation Assay to Isolate Lipids Rafts from Soft Tissue or Cells
The arrangement in eukaryotic cell membranes of liquid-ordered states surrounded by liquid-disordered phases allows for the existence of organized membrane microdomains called rafts. Rafts play a crucial role in signal-transduction events, in lipid transport and in various internalization processes, and for all these reasons need to be purified for further characterization.
PTEN-lipid Binding Assay
The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which they attach. Then the membrane is incubated with a lipid binding protein possessing an epitope tag. The protein binds to the lipid which is detected by immunoblotting with an antibody recognizing the epitope tag (see Figure 1). PTEN is an important tumor suppressor which functions as both protein and lipid phosphatase. The primary physiological substrate of PTEN is signaling lipid PtdIns (3, 4, 5) P3, by dephosphrylating PtdIns (3, 4, 5) P3 to PtdIns (4, 5) P2 PTEN negatively regulates PI3K signaling and mediates its tumor-suppressor function by inactivating downstream oncogenic AKT-mediated signaling. The PTEN lipid binding assay is conducted to study the specific binding of PTEN to different lipid molecules.
Cancer Biology
Quantitative Methylation Specific PCR (qMSP)
Detection of low copies of methylated DNA targets in clinical specimens is challenging. The quantitative Methylation-Specific PCR (qMSP) assays were designed to specifically amplify bisulphite-converted methylated DNA target sequences in the presence of an excess of unmethylated counterpart sequences. These qMSP assays are real-time PCR assays utilizing, sequence-specific primers and an intervening, also sequence specific, Taqman probe to cover an amplicon of approximately 100 bp in length. The use of Taqman probes bearing a minor groove binding (MGB) allow for the use of shorter probes and therefore facilitate design and significantly increases the analytical specificity of the reaction. In the context of the biomarker discovery program of the Liverpool Lung Project (LLP), ten gene promoters were selected. qMSP assays were developed, validated and used to screen 655 bronchial washings from patients with lung cancer and age/sex matched controls with non malignant lung disease (Nikolaidis et al., 2012).
Natural Killer Cell Transfer Assay
Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the adoptive transfer of purified NK cells into NK cell-deficient tumor bearing mice in order to establish the intrinsic functionality of NK cells.
Cell Biology
Cell Surface Protein Biotinylation and Analysis
A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control).
Sodium Current Measurements in HEK293 Cells
This protocol is used to measure sodium currents from heterologously transfected cells lines, such as HEK293 cells. Standard whole cell patch clamp technique is used to assess ion channel function. This protocol has been used to study cardiac type Nav1.5 sodium channel, and in particular to compare wild-type and mutant channels related to Brugada Syndrome (BrS). Mutations related to BrS provoke a loss of function of the cardiac sodium channel. This is evidenced by a reduction of sodium current density and/or alterations of the activation or inactivation kinetic parameters, such as a slow recovery from inactivation. These effects could explain the alteration of the cardiac action potential that leads to the characteristic ST elevation observed in the electrocardiogram of the Brugada Syndrome patients. This protocol, with some variations, is suitable for studying other cardiac arrhythmias related to alterations in the cardiac type sodium channel function as well as for studying other voltage-dependent sodium channels.
Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes
Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This protocol was used to map genes (Sharakhova et al., 2010) and microsatellite markers (Kamali et al., 2011; Peery et al., 2011) on polytene chromosomes from ovarian nurse cells and salivary glands of malaria mosquitoes. Detailed physical genome mapping performed on polytene chromosomes has the potential to link DNA sequences to specific chromosomal structures such as heterochromatin (Sharakhova et al., 2010). This method also allows comparative cytogenetic studies (Sharakhova et al., 2011; Xia et al., 2010), and reconstruction of species phylogenies (Kamali et al., 2012).
Immunology
Bronchoalveolar Lavage and Lung Tissue Digestion
Bronchoalveolar lavage (BAL) is a simple but valuable and typically performed technique commonly used for studying the pathogenesis of lung diseases such as asthma and COPD. Cell counts can be combined with new methods for examining inflammatory responses, such as ELISA, Flow cytometric analysis, immunohistochemistry, quantitative polymerase chain reaction, and HPLC to assess cellular expression for inflammatory cytokines and growth factor. Here we describe a basic procedure to collect BAL fluid and digest lung tissue for assessing a number of pulmonary components.
Ex vivo Natural Killer Cell Cytotoxicity Assay
Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the “gold standard” chromium release assay for measuring the target cell killing capacity of NK cells. Key features of this cytotoxicity assay are that it is performed with sorted NK cells as the effectors and any Major Histocompatibility Class I (MHC-I)-low or deficient tumor cell line can be used as the target cells.
Microbiology
Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans
Peptidoglycan hydrolases or autolysins are enzymes capable of cleaving covalent bonds in bacterial peptidoglycan cell wall layer. They can participate in the cell division process, in the release of turnover products from peptidoglycan during cell growth, and in cell autolysis induced under particular conditions. The protocol for zymogram presented below should enable the identification of such enzymes through their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing bacterial cells as substrate.
Drug Sensitivity Assay of Xanthomonas citri subsp. citri Using REMA Plate Method
Resazurin Microtiter Assay (REMA) is a simple, rapid, reliable, sensitive, safe and cost-effective measurement of cell viability. Resazurin detects cell viability by converting from a nonfluorescent dye to the highly red fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth (Palomino et al., 2002). The REMA assay can be used as a fluorogenic oxidation-reduction indicator in a variety of cells, including bacteria, yeast and eukaryotes (Silva et al., 2013).
![Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-3H]dihydrosphingosine](https://en-cdn.bio-protocol.org/imageup/arcimg/20130820012439267.jpg?t=1758534032)
Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-3H]dihydrosphingosine
Yeast sphingolipids can be metabolically labeled with exogenously added radioactive precursors. Here we describe a method to label ceramides and complex sphingolipids of Saccharomyces cerevisiae with a radioactive ceramide precursor, D-erythro-[4, 5-3H]dihydrosphingosine. This protocol is used to study the biosynthesis, transport and metabolism of sphingolipids in yeast.
Bioassay to Screen Pathogenesis-associated Genes in Alternaria brassicicola
Alternaria brassicicola is a necrotrophic fungus that causes black spot disease of most plants in the Brassicaceae, including cultivated Brassica species and weedy Arabidopsis species. Since the concept of transformation constructs of linear minimal elements was developed (Cho et al., 2006), we have produced over 200 strains of loss-of-function mutants with an aid of selectable marker genes. Pathogenicity assays are a time-consuming step in screening pathogenesis-associated genes among targeted gene mutants. Here we describe a method for pathogenesis assays of A. brassicicola. Using this method, we have discovered pathogenesis-associated genes and were able to further characterize the functions of selected gene (Cho et al., 2013; Cho et al., 2012; Srivastava et al., 2012).
Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells
The following protocol describes the isolation of radioactive viral and subviral particles from infected cells. This protocol has been written for isolation of poliovirus particles from H1 HeLa cells. Infection protocol and timing of [35S] methionine labeling and particle collection should be tailored to the virus of interest. Following isolation of the viral particles, the viral proteins present in these particles may be separated by gel electrophoresis and visualized by autoradiography.
Molecular Biology
Ultra-low Background DNA Cloning System
We have developed a method to clone DNA fragments into the E. coli plasmid vectors with almost 100% efficiency (Goto and Nagano, 2013). This method is based on highly efficient yeast-based in vivo cloning, and the subsequent cloning of the constructed plasmids into E. coli. Our method is useful for various applications: multifragment DNA cloning, cloning of large DNA fragments, and cloning into large plasmid vectors. Furthermore, the sites at which DNA fragments are joined are not always located at the restriction ends in the plasmid vector, thus making the cloning method more flexible. Our system does not require manipulation for assembling or joining DNA fragments in a test tube, the efficiency of which may sometimes depend on the reaction conditions or the skills of the person performing the procedure. Therefore, both success rate and efficiency are extremely high. However, our system has a disadvantage in that it requires 2 steps for transformation. Our method is an improved version of previously developed methods (Iizasa and Nagano, 2006; Nagano et al., 2007). Next figure shows the flowchart of our method.
Neuroscience
Retrograde and Anterograde Tracing of Neural Projections
Neurons consist of four elements, the soma, dendrite, axon and terminal. They work in concert as the input (soma and dendrite) and output (axon and terminal) parts of neuronal transmission. To function and maintain neuronal activity and metabolisms, proteins and organelles should be transported from soma to terminal via anterograde axonal transport, and also from terminal to soma via retrograde transport. By utilizing these transport systems, neural projection is traced by injecting tracers into local sites of interest. Furthermore, neurochemical properties, such as glutamatergic and GABAergic, can be determined by combining retrograde and anterograde tracing with fluorescent in situ hybridization and immunofluorescence.
Primary Culture of SVZ-derived Progenitors Grown as Neurospheres
SVZ-derived progenitors grown as neurospheres is a well-known model to study neural stem cell and progenitor functions such as proliferation, differentiation/self-renewal, and migration (Durbec and Rougon, 2001). This protocol is for preparing a culture of SVZ-derived progenitors from 8 early postnatal mouse brains (P0 to P3). One week after cell plating, we can observe round floating neurospheres, each resulting from the clonal expansion of a single EGF/FGF responsive neural progenitor.
Plant Science
Measuring Germination Percentage in Wheat
Knowledge of the viability of seeds is a prerequisite for establishing the seeding rate in crop production and for germination-related trait evaluation in crop plants. This method explains a simple procedure to establish germination percentage in wheat seeds.
Reverse Zymmogram Analysis for the Detection of Protease Inhibitor Activity
This protocol describes a gel-based procedure to detect protease inhibitor activity. In this method gelatin is used as a substrate for proteolysis and is copolymerized within the polyacrylamide matrix. Protein extracts are fractioned by SDS-PAGE and then the gel is treated with the protease of interest, which degrades gelatin, except in the areas where inhibitory activity is present. Inhibition of protease activity appears as colored bands against a clear background after staining with Coomassie Brilliant Blue (Figure 1). The effectiveness of the assay is dependent on the capacity of the protease inhibitor to refold after SDS-PAGE fractionation. Alternatively, it can be performed using native (PAGE) gels. Although the protocol presented here has been standardized to test for subtilisin inhibitory activity, it can easily be adapted to test other proteases and protease inhibitors.Figure 1. Protease inhibitor activity by zymogram analysis of different purified fractions and a protein crude extract. Zymogram was performed after SDS-PAGE. One microgram of protein was loaded to each lane. CE: Crude extract; QS: Fraction from anion exchange chromatography (Q-sepharose); Fractions 35-36: Obtained after a size exclusion chromatography (Superdex 200). M: Molecular markers. Arrows indicate inhibition activity.
Rapid Induction of Water Stress in Wheat
Traditional water stress evaluation studies in wheat are time consuming and can take up to several months to finish. A rapid phenotypic screening for water stress is important for accommodating time-bound water stress studies such as transient gene silencing studies in wheat. This method explains a procedure to induce water stress in young wheat plants within three weeks.
Maize Embryo Transient Transformation by Particle Bombardment
Particle bombardment has been shown to be a useful method to study gene promoter regulatory elements by transient transformation of maize embryos with different constructions of gene promoters fused to a gene reporter. DNA to transfer is coated to high density gold microparticles and introduced into cells when accelerated by a helium pulse. This method allows a first rapid approach, avoiding time consuming stable transformation of maize plants and also allows quantitative promoter expression analysis by a histochemical or fluorometric assay.
Determination of Nitrate Uptake and Accumulation Using 15N in Rice Seedlings
Nitrogen-15 is a rare stable isotope of nitrogen. This isotope is often used in agricultural research. For example, Nitrogen-15 is used to trace mineral nitrogen compounds and translocate the nitrogen molecule in plants. This protocol is used to determine nitrate uptake and accumulation in rice seedlings by using Nitrogen-15.
Measurement of Net NO3- Flux in Rice Plants with the SIET System
SIET (scanning ion-electrode technique) is a new technique to study the flow rate of the ions and molecules in real time in living biomaterials by using microelectrodes and microsensor. This technique allows non-invasive, simultaneous measurement of fluxes of specific ions at the surface of an intact plant. It has high temporal and spatial resolutions. This protocol uses the SIET system for the measurement of ions flux rate in rice plants.