Journal of Biological Methods (JBM)
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163 research outputs found
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Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation
No full textLocal stimulation with optical tweezers has been used to mimic natural stimuli that occur in biological processes such as cell migration or differentiation. Carriers (beads and lipid vesicles) with sizes down to 30 nm can be manipulated with a high spatial and temporal resolution: they are positioned with a sub-micrometric precision on a specific cell compartment and the beginning of the stimulation can be triggered with millisecond precision. RhoGTPases are a Ras-related family of proteins that regulate many different functions including cell polarity, microtubule dynamics and membrane transport pathways. Here we combine local stimulation with FRET microscopy to study RhoGTPases spatial and temporal activation following guidance cue local stimulation. We used two different vectors for local delivery: silica micro-beads and micro-sized lipid vesicles. The experimental methods associated with neuronal growth cone local stimulation are discussed in detail, as well as the analysis methods. Here we present a protocol that enables to study neuronal growth cone cytoskeleton rearrangements in response to a gradient of molecules in a way that better mimics physiological conditions, and it can be similarly applied to each secreted molecule involved in cell signaling
A guide to simple, direct, and quantitative in vitro binding assays
No full textRecent advances in proteomic screening approaches have led to the isolation of a wide variety of binding partners to interacting proteins and opened an avenue to analyze and understand signaling pathways. The study of protein-protein interactions is a key component in elucidating and understanding signaling pathways. Despite the importance of these interactions, very few studies are quantitative or report binding affinities. Here we present a simple method for examination and analysis of direct protein-protein binding interactions between two purified proteins. In the quantitative pull-down assay, one protein (the bait protein) is immobilized on beads whereas a second protein (the prey) is kept in solution. The concentration of the bait protein is kept constant, whereas the concentration of the prey protein is increased until binding saturation is achieved. After incubation, the beads are precipitated to separate unbound prey protein in solution from prey protein bound to the bait. The fraction of bound prey protein can then be loaded on a protein gel and the resulting bands can be analyzed with standard software. The quantitative pull-down assay with purified recombinant proteins provides a simple method to obtain dissociation constants (Kd). These quantifications are invaluable to compare relative binding of proteins, to map binding sites, and to show that binding is direct. This assay presents a powerful method to quantitatively analyze protein-protein interactions with tools that are available in most biochemistry laboratories and does not require the use of specialized or expensive equipment
Easy and cost-effective stable positioning of suspension cells during live-cell imaging
No full textDynamic processes of cells can be best monitored when living cells are analyzed by imaging. While it is easy to observe adherent living cells it has been extremely challenging to analyze suspension cells. This cell type floats freely in the culture dish, and it is only a question of time when the focus or the observation field is lost. In order to keep the cells in focus, an easy and inexpensive method allowing the observation of living suspension cells during confocal laser scanning microscope imaging was developed
Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
No full textProtein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and dephosphorylation by monitoring migration on high resolution peptide gels. In this study, we demonstrate the utility of such a technique using small peptides corresponding to cyclin-dependent kinase-1 (Cdk1)/cyclin B1 sites in two important apoptotic regulatory proteins, Bcl-xL and caspase-9. We show that the mobility of the peptides is retarded following Cdk1-mediated phosphorylation, and that peptide dephosphorylation, catalyzed either by purified phosphatase or by crude cell extracts, is readily observable by increased peptide gel mobility. Furthermore, the procedure can be conducted without the use of radioactive adenosine triphosphate (ATP), and does not require any specialized reagents or apparatus. The method can be used to identify and characterize specific kinase and phosphatases responsible for phosphorylation and dephosphorylation of specific sites in any protein of interest
A method for evaluating cocaine-induced social preference in rats
No full textDrug addicts are extremely sensitive to cues that predict drug availability and exposure to these cues can facilitate drug relapse. Cues vary in their nature but can include drug-associated paraphernalia, environmental contexts, and discrete conditioned stimuli (e.g., advertisements). One cue that has recently been heavily investigated is that of social interaction. To date, it has been demonstrated that when cocaine is conditioned with social interaction, place preference for cocaine significantly increases, suggesting that the presence of social interaction during a drug-associated “high” enhances the magnitude of drug reward. When social interaction is provided in a mutually exclusive, non-drug environment though, it can serve as a preventative stimulus towards cocaine seeking. What remains unknown is whether contact with rats associated with drug experience facilitates preferential social interactions for those rats. The first step in answering this question is to determine if rats can behaviorally discriminate between drug-associated and non-drug-associated conspecifics, much like humans can differentiate their “drug-friends” from their non-drug-using friends. Using a custom social interaction chamber, in which rats were able to interact with two distinct conspecifics via holes in a boundary wall, we demonstrate that rats exhibit more interactive and investigative behavior towards a partner that was consistently present during the drug-state, than a partner that was present when the rat was “sober”. It is our hope that this protocol will contribute to the development of models designed to study social cue-induced reinstatement, and related neural substrates, and will ultimately contribute to the treatment of substance use disorders
RATA: A method for high-throughput identification of RNA bound transcription factors
No full textLong non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-boundtranscription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest. RATA requires only ~5 million cells and standard molecular biology reagents for multiplexed identification of up to 96 transcription factors in 2–3 d. Thus, RATA offers significant advantages over other technologies for analysis of RNA-transcription factor interactions
Total proteome turbidity assay for tracking global protein aggregation in the natural cellular environment
No full textProteome homeostasis is crucial for optimal cellular function and survival in the face of various stressful impacts. This entails preservation of a balance between protein synthesis, folding, degradation, and trafficking collectively termed proteostasis. A hallmark of proteostasis failure, which underlies various diseases, is enhanced misfolding and aggregation of proteins. Here we adapted the measurement of protein turbidity, which is commonly used to evaluate aggregation of single purified proteins, for monitoring propensity for aggregation of the entire soluble cellular proteome incubated in vitro for several hours. We show that over-expression of an aggregation-prone protein or applying endoplasmic-reticulum (ER) stress to either cells in culture or to the intact organism, Drosophila, enhances the rise in turbidity of the global soluble proteome compared to untreated cells. Additionally, given that Alzheimer’s disease (AD) is known to involve ER stress and aggregation of proteins, we demonstrate that the soluble fraction of brain extracts from AD patients displays markedly higher rise of global proteome turbidity than in healthy counterparts. This assay could be valuable for various biological, medical and biotechnological applications
Resolving biofilm topography by native scanning electron microscopy
No full textScanning electron microscopy (SEM) is a powerful tool for structural analysis, but it requires biological samples to undergo lengthy, chemically-complex multi-step preparation procedures, arguably altering some features in the sample. Here we report an ultra-rapid and chemical-free technique for visualizing bacterial biofilms at their native state. Our technique minimizes the time interval from culture to imaging to approximately 20 min, while producing high-resolution images that enable the detection of a variety of topographic features such as bacterial chains, and resolving cells from matrix. We analyzed images obtained from Bacillus subtilis biofilms, demonstrate the usefulness of this technique for multiple types of image analysis, and discuss its potential to be improved and adapted to other types of biological samples
A cost-effective RNA extraction technique from animal cells and tissue using silica columns
No full textRibonucleic acid (RNA) is widely used in molecular biology assays, and some of the most common assays include: northern blotting and RT-PCR gene expression analysis. RNA is generally extracted by two methods: phenol-chloroform or commercially available silica spin column kits. Phenol-chloroform extraction is generally more economical; however, it produces hazardous byproducts, and leftover chemicals in the sample that can inhibit downstream applications. Commercial kits usually have simple set ups and short preparation time; however, they can introduce a significant expense to laboratory budgets. Here we have created a method to extract RNA using generic silica columns and readily available reagents while maintaining a high yield and purity
Using cluster edge counting to aggregate iterations of centroid-linkage clustering results and avoid large distance matrices
No full textSequence clustering is a fundamental tool of molecular biology that is being challenged by increasing dataset sizes from high-throughput sequencing. The agglomerative algorithms that have been relied upon for their accuracy require the construction of computationally costly distance matrices which can overwhelm basic research personal computers. Alternative algorithms exist, such as centroid-linkage, to circumvent large memory requirements but their results are often input-order dependent. We present a method for bootstrapping the results of many centroid-linkage clustering iterations into an aggregate set of clusters, increasing cluster accuracy without a distance matrix. This method ranks cluster edges by conservation across iterations and reconstructs aggregate clusters from the resulting ranked edge list, pruning out low-frequency cluster edges that may have been a result of a specific sequence input order. Aggregating centroid-linkage clustering iterations can help researchers using basic research personal computers acquire more reliable clustering results without increasing memory resources