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Past Issue in 2018
Volume: 8, Issue: 1
Biochemistry
Determining Ribosome Translational Status by Ribo-ELISA
The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules that are isolated by physical fractionation in sucrose gradients or other methods.
Cell Biology
High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al., 2017).
Immunology
Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with pervanadate or H2O2 is detected with antibodies specific for the sulfonic acid (SO3H) form of the conserved active site cysteine of PTPs. In this protocol, we describe a method for the detection of the reduced (S-; active) or irreversibly oxidized (SO3H; inactive) form of the hematopoietic PTP SHP-1 in thymocytes, although this method is applicable to any cysteine-dependent PTP in any cell type.
Microbiology
Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9
This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.
Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins. In this case, we show that the high isoelectric point of the putative partner results in a poor transfer. Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.
Transplantation of Fecal Microbiota Shaped by Diet
Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with protection from disease. We reconstituted germ free (GF) NOD mice with fecal bacteria from protected NOD mice fed with high acetate- and butyrate-yielding diets, to test whether the transferred gut microbiota protect against the development of T1D. GF NOD mice that received a microbiota shaped by high acetate- but not butyrate-yielding diet showed a marked protection against diabetes. This fecal transplantation assay demonstrated the potential for a dietary technology to reshape the gut microbiota that enables specific bacteria to transfer protection against T1D.
Bacterial Aggregation Assay in the Presence of Cyclic Lipopeptides
Lipopeptides is an important class of biosurfactants having antimicrobial and anti-adhesive activity against pathogenic bacteria. These include surfactin, fengycin, iturin, bacillomycin, mycosubtilin, lichenysin, and pumilacidin (Arima et al., 1968; Naruse et al., 1990; Yakimov et al., 1995; Steller and Vater, 2000; Roongsawang et al., 2002; Vater et al., 2002). To date, none of these lipopeptides have been reported to possess any anti-motility activity. We isolated, purified and characterized two novel cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. CLPs dramatically suppress the motility of pathogenic bacterium Vibrio alginolyticus 178, and promote cellular aggregation without inducing cell death. Cell aggregation assay was performed with the modification according to methods described by Dalili for anti-biofilm assay (Dalili et al., 2015). In future, this assay can be adapted to test both the cell aggregation and anti-biofilm activity of lipopeptide-like active substances derived from bacteria.
Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER
Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and direct usage of the permeabilized cells for the determination of metabolic enzyme activities in vivo.
Neuroscience
The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.
Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can be visualized by directly observing the beads bearing immunoprecipitates under a fluorescence microscope. This assay can examine a large number of protein combinations at one time, without requiring time-consuming procedures, including SDS-PAGE and immunoblotting. Furthermore, the VIP assay can examine complicated one-to-many and many-to-many protein interactions. Another important point of the VIP assay is the use of nanobodies for immunoprecipitation. A Nanobody is a single-domain antibody derived from Camelidae (camels and relatives). Because of its small size, high-affinity, high-specificity, and stability, anti-GFP Nanobody expressed in E. coli can be purified on a large scale, and used virtually inexhaustibly for immunoprecipitation experiments. Here we describe protocols for preparation of GST-tagged anti-GFP Nanobody and the VIP assay.
A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats
Permanent occlusion of bilateral common carotid arteries (2VO) in rat is considered as a suitable animal model to mimic chronic brain hypoperfusion status, which is proved to be a risk factor to precede the Alzheimer’s disease or/and vascular dementia. In this protocol, we describe how to successfully ligate the bilateral common carotid arteries covered by anterior cervical muscle group, and provide the details for understanding the surgical procedures of 2VO.
Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration.
Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in Caenorhabditis elegans
Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a C. elegans population behavioral assay used to measure locomotory responses to changes in environmental oxygen (O2) and carbon dioxide (CO2) concentrations. These behavioral assays are high-throughput and enable examination of genetic, neuronal and circuit function.
Plant Science
In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6
RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.
Micro-computed Tomography to Visualize Vascular Networks in Maize Stems
Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the tissue. Here we describe a protocol to utilize micro-computed tomography (micro-CT) scanning to visualize vascular networks in the maize stem. The protocol covers sample fixation and staining with contrasting reagents, data acquisition using micro-CT, reconstructing three-dimensional (3D) models of stem inner structures and extraction of vascular networks from the model. This protocol can be easily applied to various types of species and organs/tissues.
Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes
Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses (Bömer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel badnavirus genomes.
Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues–from High Pressure Freezing Preparation to Ultrathin Section Collection
Plasmodesmata (PD) are nanometric (~20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from cell-to-cell and then systematically throughout the whole plant. Their central position in plant biology makes it crucial to understand their physiology and especially link their function to their structure. Over the past 50 years, electron microscopists have observed them and attempted to ultrastructurally characterize them. They laid the foundation of what is known about these pores (Tilney et al., 1991; Ding et al., 1992; Oparka and Roberts, 2001; Nicolas et al., 2017a).Despite the explosion of three-dimensional electron microscopy (3D-EM), PD ultrastructure remained recalcitrant to such technique. The first technical difficulty is to process them in such a way where they are as close to their native state as possible. Secondly, plant samples reveal themselves as being difficult to process due to the poor staining/fixating reagents penetration rates, their increased size, their high water content and the presence of an acidic vacuole. On top of this, their very unique position in the cell wall and their nanometric size make them difficult to conveniently stain in order to see the inner-workings of these pores.Here we describe in detail the protocol used in Nicolas et al. (2017b) to image PD in fine detail and produce high-resolution tomograms.
Real-time Analysis of Auxin Response, Cell Wall pH and Elongation in Arabidopsis thaliana Hypocotyls
The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH.
Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri
Triacylglycerols (TAGs) are esters formed from one glycerol and three fatty acids. TAGs are induced to accumulate in algal cells under environmental stress conditions including nutrient-limitation, hyperosmosis, and low temperature, for the storage of metabolic energy and carbon, and also for the consumption of excess energy (e.g., Hirai et al., 2016; Hayashi et al., 2017). Beside their physiological significance, the commercial utilization of algal TAG has been expected for the production of biodiesel, the methyl esters of fatty acids, from the aspect of carbon-neutral conception. The amounts of TAGs can be determined through quantitative measurement of their constituent fatty acids. This protocol consists of the following three parts: the first is the extraction of total lipids from algal cells with the use of organic solvents, chloroform and methanol, according to the method of Bligh and Dyer (1959), the second is the separation of TAG from the other lipid classes by thin-layer chromatography (TLC), and the third is the production of methyl-esterified derivatives of their constitutive fatty acids and subsequent quantitation of them by capillary gas-liquid chromatography (GLC). This protocol adapted from Sato and Tsuzuki (2011) is used for TAG analysis in a green alga, Chlorella kessleri.
Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System
Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster (Mito et al., 2005), but the presented protocol has been developed specifically for Arabidopsis thaliana (Sura et al., 2017).