Improve Research Reproducibility A Bio-protocol resource
- Submit a Protocol
- Receive Our Alerts
- EN
- Protocols
- Articles and Issues
- For Authors
- About
- Become a Reviewer
Past Issue in 2015
Volume: 5, Issue: 14
Biochemistry
Nanofluidic Proteomic Immunoassay
Nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, has the potential to quantitatively characterize protein post-translational modifications that cause shifts in isoelectric point (pI). This protocol details the NIA analysis of protein phosphorylation using AKT as an example. This protocol can be used for two platforms, NanoPro 1000 and Peggy Sue, from ProteinSimple Company. NanoPro 1000 separates proteins based on charge. Peggy Sue separates proteins based on either charge or size. The platforms can analyze up to 96 samples at a time with lysates from as few as 25 cells per assay. Detailed information of the platforms is available on ProteinSimple’s website (http://www.proteinsimple.com).
Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology
Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck that slows down the structural biology of IMPs is the preparation of diffracting crystals or concentrated solutions of stable isotope labeled IMPs. Here, we describe procedures to monitor the quality of aqueous solutions of [2H, 15N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of β-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with α-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al., 2013). 2D [15N, 1H]-correlation maps are used as “fingerprints” to assess the foldedness of the IMP in solution. For promising samples, these “inexpensive” data are then supplemented with measurements of the translational and rotational diffusion coefficients, which give information on the shape and size of the IMP/detergent mixed micelles. Using microcoil equipment for these NMR experiments enables data collection with only micrograms of protein and detergent. This makes serial screens of variable solution conditions viable, enabling the optimization of parameters such as the detergent concentration, sample temperature, pH and the composition of the buffer.
Cell Biology
A Simple Method to Generate Gene Knockout Clones in Human Cells Using Transcription Activator-Like Effector Nuclease (TALEN)
Transcription activator-like effectors (TALEs) are naturally occurring proteins secreted by the plant pathogen, Xanthomonas, and fused to the Fok1 endonuclease to generate TALE nucleases (TALENs). TALEN pairs bind to specific DNA sequences initiating Fok1 dimerization and double-stand cleavage of DNA within the TALEN target site. This cleavage event triggers cellular repair mechanisms that result in insertions and/or deletions (indels), which enable gene knockout. The high specificity and efficiency of TALENs makes them important tools for genome editing. Here, we describe a method for the generation of single-cell clones with targeted gene knockout by TALEN using co-transfection and FACS with a fluorescent reporter. This protocol was designed to knockout cell death-inducing DFFA-like effector b, CIDEB, in Huh7.5 cells; however, this protocol can be applied to a wide range of cell types and genes of interest.
Microbiology
Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata
Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014).
Genetic Transformation of Candida glabrata by Heat Shock
Here, we report a method for the transformation of Candida glabrata using a heat shock method. The protocol can be used for transformations in single well or in 96-well scale. It has been employed as an alternative method to the electroporation protocol to construct a genome-scale gene deletion collection in the human fungal pathogen Candida glabrata ATCC2001 and related strains. Furthermore, the protocol can be used to generate gene deletions in clinical isolates of Candida glabrata (C. glabrata).
Generation of Mammalian Host-adapted Leptospira interrogans by Cultivation in Peritoneal Dialysis Membrane Chamber Implantation in Rats
Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti et al., 2003; Ko et al., 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize the proximal convoluted renal tubules (Athanazio et al., 2008). Infected hosts shed large number of spirochetes in their urine and the leptospires can survive in different environmental conditions before transmission to another host. Differential gene expression by Leptospira spp. permits adaption to these new conditions. Here we describe a protocol for the cultivation of Leptospira interrogans within Dialysis Membrane Chambers (DMCs) implanted into the peritoneal cavities of Sprague-Dawley rats (Caimano et al., 2014). This technique was originally developed to study mammalian adaption by the Lyme disease spirochete, Borrelia burgdorferi (Akins et al., 1998; Caimano, 2005). The small pore size (8,000 MWCO) of the dialysis membrane tubing used for this procedure permits access to host nutrients but excludes host antibodies and immune effector cells. Given the physiological and environmental similarities between DMCs and the proximal convoluted renal tubule, we reasoned that the DMC model would be suitable for studying in vivo gene expression by L. interrogans. In a 20 to 30 min procedure, DMCs containing virulent leptospires are surgically-implanted into the rat peritoneal cavity. Nine to 11 days post-implantation, DMCs are explanted and organisms recovered. Typically, a single DMC yields ~109 mammalian host-adapted leptospires (Caimano et al., 2014). In addition to providing a facile system for studying the transcriptional and physiologic changes pathogenic L. interrogans undergo within the mammal, the DMC model also provides a rationale basis for selecting new targets for mutagenesis and the identification of novel virulence determinants.Caution: Leptospira interrogans is a BSL-2 level pathogen and known to be excreted in the urine of infected animals. Animals should be handled and disposed of using recommended Animal Biosafety Levels (ABSL) for infectious agents using vertebrate animal guidelines. Note: All protocols using live animals must conform to governmental regulations regarding the care and use of laboratory animals. The success of this protocol is dependent on the proper use of aseptic techniques during all stages of both dialysis membrane chamber preparation and animal surgery.
Genetic Transformation of Candida glabrata by Electroporation
Here, we report a method for the transformation by electroporation of the human fungal pathogen Candida glabrata (C. glabrata). The protocol can be used for transformations in single well or in 96-well microtiter plates. It has been extensively used to generate a genome-scale gene deletion library using the C. glabrata background recipient strain ATCC2001 (Schwarzmüller et al., 2014).
Molecular Biology
In vitro DNA Protection Assay Using Oxidative Stress
A wide range of stresses such as oxidative stress, acid, alkaline, UV, and metal can damage DNA. Here, we describe a protocol to measure the DNA nicking damage by Fenton reaction-mediated oxidative stress. Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH- + ∙OH) produces the highly deleterious hydroxyl radicals that damage the cellular components such as DNA, lipid and proteins.
Neuroscience
A Rat Model of Intracerebral Hemorrhage Induced by Collagenase IV
Stroke, the second leading cause of death worldwide (Ingall, 2004), is one of the major causes of morbidity and mortality. Intracerebral hemorrhage (ICH), a lethal type of stroke, accounts for 20% of all strokes (Qureshi et al., 2001), and occurs in about 50-60% of Asians (Inagawa, 2002). In order to understand the disease process, three animal model of ICH have been used to study the pathophysiology and treatment of ICH, including the microballoon model, the bacterial collagenase injection model (Rosenberg et al., 1990) and the autologous blood injection model (Andaluz et al., 2002; Belayev et al., 2003). In the collagenase injection model, the hemorrhage size is controllable which was induced by small vessel breakdown. This model also can mimic the onset of spontaneous intraparenchymal bleeding and the expansion of continuous bleeding in ICH patients (Kazui et al., 1996; MacLellan et al., 2008; James et al., 2008). In the past several years, our previous studies have proven that our modified collagenase IV injection model is a reliable and reproducible model of ICH in rat (Lu et al., 2014; Gao et al., 2014; Wang et al., 2011). We hereby introduce our model of ICH as following.
Plant Science
Circular RT-PCR Assay Using Arabidopsis Samples
Post-transcriptional processing is critical for RNA biogenesis, in which conventional functional RNA transcripts are generated, such as messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) for translation as well as emerging non-coding RNAs with known or unknown regulatory functions. To determine the precise termini of an RNA molecule during or after processing, the primer extension and Rapid Amplification of cDNA Ends (RACE) methods have been routinely utilized for the precise mapping of 5’ or 3’ ends. Different from these assays, which are designed to detect only one end of a specific target RNA at a time, circular Reverse Transcription-Polymerase Chain Reaction (cRT-PCR) is able to simultaneously determine both the 5’ and 3’ ends of the target RNA. In Arabidopsis thaliana, cRT-PCR has been wildly applied to identify both the 5’ and 3’ extremities of the ribosomal RNA precursors, or to assess the length or post-transcriptional extensions at the 3’ end of a matured mRNA. In this protocol, we summarize and present a detailed procedure of the cRT-PCR assay in Arabidopsis thaliana, which is also successfully used in our previously published work (Hang et al., 2014).
Chlorophyll Fluorescence Measurements in Arabidopsis Wild-type and Photosystem II Mutant Leaves
Chlorophyll fluorescence measurement is a widely used technique to determine photosynthetic performance. Light energy absorbed by a chlorophyll molecule can be dissipated by driving photochemical energy conversion, as heat in non-photochemical quenching processes, or it is re-emitted as fluorescence. The loss of light energy as chlorophyll fluorescence is primarily derived from photosystem II. Photosystem II is a thylakoid-embedded multiprotein complex which provides the high redox potential needed to oxidize water. Within photosystem II photons of light are captured and used to energize electrons. Energized electrons are fed into the linear electron transport chain and photosystem II replenishes lost electrons with electrons from splitting of water. Chlorophyll fluorescence yield can be quantified using a modulated fluorometer device. In such a device, a modulated measuring light beam (switched on and off at a high frequency) and the parallel detection of fluorescence exclusively excited by the measuring light allows chlorophyll fluorescence measurements in the presence of photosynthetic (actinic) light. In addition, high intensity, but short duration light flashes (saturating pulses) are used to determine maximum fluorescence yields in dark and light adapted leaves. In this protocol the procedure to receive a typical fluorescence graph of Arabidopsis wild-type leaves is given. Furthermore, this procedure can be used to identify Arabidopsis mutant plants affecting photosystem II, on the basis of the respective fluorescence graphs and values.
A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten
Protein phosphorylation is one of the most common post-translational modifications in eukaryotic cells and plays a critical role in a vast array of cellular processes. Efficient methods of protein extraction and phosphopeptide purification are required to ensure the detection of high quality of proteins. In our hands, phenol extraction of proteins and TiO2 chromatography enrich phosphorylated peptides more efficiently than other methods in the moss Physcomitrlla patens (P. patens).
Atomic Force Microscopy (AFM) Analysis of Cell Wall Structural Glycoproteins in vitro
Hydroxyproline-rich glycoproteins (HRGPs) are major protein components in dicot primary cell walls and generally account for more than 10% of the wall dry weight. As essential members of the HRGP superfamily, extensins (EXTs) presumably function in the cell wall by assembling into positively charged protein scaffolds (Cannon et al., 2008) that direct the proper deposition of other wall polysaccharides, especially pectins, to ensure correct cell wall assembly (Hall and Cannon, 2002; Lamport et al., 2011a). Extensins are recalcitrant to purification as they are rapidly cross-linked into a covalent network after entering the cell wall but there exists a short time window in which newly synthesized extensin monomers can be extracted (Smith et al., 1984; Smith et al., 1986) by salt elution. A detailed protocol for extraction of extensin and other wall structural proteins has been described earlier (Lamport et al., 2011b). The protocol elaborated here provides an approach to studying the self-assembly of extensins and potentially of other cell wall components in vitro using AFM.
Phakopsora pachyrhizi Infection Bioassay in Detached Soybean Transgenic Leaves for Candidate Gene Validation
Soybean (Glycine max) is one of the most important crops in the world. Phakopsora pachyrhizi is a plant pathogenic basidiomycete fungus that infects soybean, causing Asian Soybean Rust (ASR) disease and affecting production. Here, we describe how to prepare the plant material and the uredospore suspension (from spores harvested from leaves exhibiting sporulating uredinia) for in vitro leaf infection. Plant material is sprayed with the uredospore suspension and incubated for 12 days. During the incubation period, the presence of lesions and pustules is visually verified. After this incubation period, the leaves are classified according to the lesion type. The number of uredospores per CM2 of leaf was also estimated. The detached-leaf assay is routinely used to test fungicide efficiency (Scherb and Mehl, 2006). Detached-leaf, greenhouse and field results have been shown to be significantly correlated (Twizeyimana et al., 2007). The present protocol was adapted from the two publications cited above. The usefulness of this approach for studying P. pachyrhyzi infection on transgenic soybean was previously demonstrated by our research team (Wiebke-Strohm et al., 2012; Bencke-Malato et al., 2014).
An Evaluation of Cellulose Degradation Affected by Dutch Elm Disease
The pathogenic fungus Ophiostoma novo-ulmi spreads within the secondary xylem vessels of infected elm trees, causing the formation of vessel plugs due to tyloses and gels, which ultimately result in Dutch elm disease. Foliage discoloration, wilting and falling from the tree are typical external leaf symptoms of the disease followed by the subsequent death of sensitive trees. Cellulolytic enzymes produced by the fungus are responsible for the degradation of medium molecular weight macromolecules of cellulose, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels but rarely in the fibers (Ďurkovič et al., 2014). The goal of this procedure is to evaluate the extent of cellulose degradation by a highly aggressive strain of O. novo-ulmi ssp. americana × novo-ulmi. Size-exclusion chromatography (SEC) compares molecular weight distributions of cellulose between the infected and the non-infected elm trees, and reveals changes in the macromolecular traits of cellulose, including molecular weights, degree of polymerization, and polydispersity index. 13C magic angle spinning nuclear magnetic resonance (13C MAS NMR) spectra help to identify and also to quantify the loss of both crystalline and non-crystalline cellulose regions due to degradation. The procedure described herein can also be easily used for other woody plants infected with various cellulose-degrading fungi.