Protocols in Current Issue
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0 Q&A 690 Views Nov 20, 2022

Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions.

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0 Q&A 2495 Views Nov 5, 2020
The Xenopus oocyte is a powerful system for the exogenous expression and functional characterization of plant membrane transport proteins. Until now, a number of potassium transporters and channels have been identified in oocytes expression system by the two-electrode voltage clamp technology. It is difficult to characterize K+/H+ anti-transporters, especially, electroneutral transporter. The K+ efflux assay system enables easy, fast, large-scale measurement of the transporters activity without two-electrode voltage clamp technology. This protocol describes a technique to measure the efflux activity of potassium transporter in oocytes expressing system.
0 Q&A 2676 Views Oct 20, 2020
Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various types of cancers. The untargeted analysis contributes to finding new biomarkers. i.e., the analysis of all detectable signals including both known and unknown metabolites extends the coverage of metabolite to be observed. However, the observed data includes thousands of peaks. Besides, non-linear migration time fluctuation and skewed peaks are caused by the sample condition. The presented pretreatment protocols of saliva samples enhance the reproducibility of migration time drift, which facilitates the matching peaks across the samples and also results in reproducible absolute concentrations of the detected metabolites. The described protocols are utilized not only for saliva but for any liquid samples with slight modifications.
0 Q&A 2309 Views Aug 20, 2020
The yeast Saccharomyces cerevisiae has been perceived over decades as a highly valuable model organism for the investigation of ion homeostasis. Indeed, many of the genes and biological systems that function in yeast ion homeostasis are conserved throughout unicellular eukaryotes to humans. In this context, measurement of the yeast cellular ionic content provides information regarding yeast response to gene deletion or exposure to chemicals for instance. We propose here a protocol that we tested for the analysis of 12 elements (Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, K+, Mg2+, Mn2+, Na+, Ni2+, Zn2+) in yeast using Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). This technique enables determination of the cellular content of numerous ions from one biological sample.
0 Q&A 4555 Views Apr 5, 2020
Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from titration series without a need to label or immobilize one or more interaction partners. Furthermore, second messenger homeostasis involving Ca2+-ions requires in particular knowledge about stoichiometries and affinities of Ca2+-binding to Ca2+-sensor proteins or Ca2+-dependent regulators, which can be obtained by employing ITC. We used ITC to measure these parameters for a set of neuronal Ca2+-sensor proteins operating in photoreceptor cells. Here, we present a step wise protocol to (a) measure Ca2+ interaction with the Ca2+-sensor guanylate cyclase-activating protein 1, (b) to design an ITC experiment and prepare samples, (c) to remove Ca2+ nearly completely from Ca2+ binding proteins without using a chelating agent like EGTA.
0 Q&A 2286 Views Mar 20, 2020
Rats are highly social animals, and mainly communicate with one another in two ways: through ultrasonic vocalizations and pheromones. Most research on pheromones has been dedicated those regarding sexual behavior, but more recently pheromones which signal danger to conspecifics have been identified in rodents. In fact, rats are capable of communicating information regarding a specific fear to a companion with which they share a cage. If a rat is trained to associate a previously neutral odor with a foot shock and then pair housed with another rat, the companion will also display a fear response specific to the trained odor, despite never being shocked itself. This communication relies on pheromones; presenting soiled bedding from a shocked rat to an individually housed naïve rat produces the same fear response in the naïve rat. The current protocol describes how to produce this phenomenon in adult Sprague Dawley rats. It is simple and easily reproduced, requires minimal equipment, and may be completed within one week.
0 Q&A 5249 Views Feb 5, 2020
Calcium (Ca2+) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca2+]) in different pathophysiological conditions. Ca2+ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex subcellular compartmentalization.

Many Ca2+ sensors are presently available (chemical and genetically encoded) that can be specifically targeted to different cellular compartments. Using these probes, it is now possible to monitor Ca2+ dynamics of living cells not only in the cytosol but also within specific organelles. The choice of a specific sensor depends on the experimental design and the spatial and temporal resolution required.

Here we describe the use of novel Förster resonance energy transfer (FRET)-based fluorescent Ca2+ probes to dynamically and quantitatively monitor the changes in cytosolic and mitochondrial [Ca2+] in a variety of cell types and experimental conditions. FRET-based sensors have the enormous advantage of being ratiometric, a feature that makes them particularly suitable for quantitative and in vivo applications.
0 Q&A 4559 Views Apr 5, 2019
We describe a protocol to measure the contribution of humidity on cell death during the effector-triggered immunity (ETI), the plant immune response triggered by the recognition of pathogen effectors by plant resistance genes. This protocol quantifies tissue cell death by measuring ion leakage due to loss of membrane integrity during the hypersensitive response (HR), the ETI-associated cell death. The method is simple and short enough to handle many biological replicates, which improves the power of test of statistical significance. The protocol is easily applicable to other environmental cues, such as light and temperature, or treatment with chemicals.
0 Q&A 5155 Views Sep 5, 2018
There is a pressing need to develop sustainable and efficient methods to protect and stabilize iron objects. To develop a conservation-restoration method for corroded iron objects, this bio-protocol presents the steps to investigate reductive dissolution of ferric iron and biogenic production of stabilizing ferrous iron minerals in the strict anaerobe Desulfitobacterium hafniense (strains TCE1 and LBE). We investigated iron reduction using three different Fe(III) sources: Fe(III)-citrate (a soluble phase), akaganeite (solid iron phase), and corroded coupons. This protocol describes a method that combines spectrophotometric quantification of the complex Fe(II)-Ferrozine® with mineral characterization by scanning electron microscopy and Raman spectroscopy. These three methods allow assessing reductive dissolution of ferric iron and biogenic mineral production as a promising alternative for the development of an innovative sustainable method for the stabilization of corroded iron.
1 Q&A 8274 Views Apr 5, 2018
The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

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