Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.The Use of Dansyl-Calmodulin to Study Interactions with Channels and Other Proteins
Steady-state fluorescence spectroscopy is a biophysical technique widely employed to characterize interactions between proteins in vitro. Only a few proteins naturally fluoresce in cells, but by covalently attaching fluorophores virtually all proteins can be monitored. One of the first extrinsic fluorescent probes to be developed, and that is still in use, is dansyl chloride. We have used this method to monitor the interaction of a variety of proteins, including ion channels, with the Ca2+-dependent regulatory protein calmodulin. Here we describe the preparation and use of dansyl-calmodulin (D-CaM). (Source: Springer ...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Förster Resonance Energy Transfer-Based Imaging at the Cell Surface of Live Cells
Understanding the molecular mechanisms of protein–protein interactions at the cell surface of living cells is fundamental to identifying the nature of cellular processes. Here, we discuss how fluorescence-based approaches have been successfully developed to visualize protein–protein interactions in living cells. Förster resonance energy transfer (FRET) is unique in generating fluorescence signals between proteins that are highly spatially sensitive. Furthermore, total internal reflectance fluorescence (TIRF) microscopy combined with FRET is a robust technique used to assay protein/protein interactions and ...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Using Total Internal Reflection Fluorescence Microscopy to Observe Ion Channel Trafficking and Assembly
Ion channels are integral membrane proteins that allow the flow of ions across membranes down their electrochemical gradients and are a major determinant of cellular excitability. They play an important role in a variety of biological processes as diverse as insulin release from beta cells in the pancreas through to cardiac and smooth muscle contraction. We have used total internal reflection fluorescence (TIRF) microscopy to watch ion channels being transported in vesicles along microtubules within the cytoplasm of the cell. Furthermore, we can directly observe the fusion of these vesicles with the plasma membrane and the...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Recording Macroscopic Currents in Large Patches from Xenopus Oocytes
The excised inside-out patch clamp technique gives rapid access to the intracellular surface of the plasma membrane while measuring channel activity. This way the effects of intracellular regulators of ion channels or transporters can be studied in isolation, in the absence of most of the cellular machinery. This chapter summarizes our experience with this technique using large patches to study various ion channels expressed in Xenopus oocytes. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Recording of Ion Channel Activity in Planar Lipid Bilayer Experiments
Planar lipid bilayer is an electrophysiological technique that enables study of functional activities of ion channels, porins, and other pore-forming molecular complexes. The main purpose of this method is to monitor ion channels’ behavior at the single molecule level in the artificial membranes. Here, I describe the details of this technique that will underline formation of the lipid bilayers and incorporation and activation of the ion channel protein. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Conventional Micropipette-Based Patch Clamp Techniques
The patch clamp technique revolutionized the study of ion channels and is considered the gold standard of measuring ion channel activity, from the academic laboratory to industrial-scale drug screening. This technique enables the study of ion channels, from single molecules up to the whole-cell ion channel population, and in their native environment. Whilst the study of single protein molecular behavior is the ultimate goal of biophysicists from all fields, this is a routine ability for ion channel specialists. This chapter is aimed at helping the beginner to design patch clamp experiments and to obtain the fundamental mic...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Two-Electrode Voltage Clamp
Two-electrode voltage clamp (TEVC) is a conventional electrophysiological technique used to artificially control the membrane potential (V
m) of large cells to study the properties of electrogenic membrane proteins, especially ion channels. It makes use of two intracellular electrodes—a voltage electrode as V
m sensor and a current electrode for current injection to adjust the V
m, thus setting the membrane potential at desired values and recording the membrane current to analyze ion channel activities. Here we describe the use of TEVC in combination with exogenous mRNA...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Recording Single-Channel Currents Using “Smart Patch-Clamp” Technique
Microdomains that form on the plasma membrane of cells are essential for signalling compartmentation within cells. The localization of ion channels in these surface microdomains is important in defining what signalling cascades will be generated. For example, in cardiomyocytes, similar to other excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. In this chapter we describe a novel advanced patch-clamp technique, “Smart patch-clamp,” which enables the study of functional ion channels in the cell surface microdomains in a...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Automated Planar Patch-Clamp
Ion channels are integral membrane proteins that regulate the flow of ions across the plasma membrane and the membranes of intracellular organelles of both excitable and non-excitable cells. Ion channels are vital to a wide variety of biological processes and are prominent components of the nervous system and cardiovascular system, as well as controlling many metabolic functions. Furthermore, ion channels are known to be involved in many disease states and as such have become popular therapeutic targets. For many years now manual patch-clamping has been regarded as one of the best approaches for assaying ion channel functi...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Piezo-Electrically Driven Mechanical Stimulation of Sensory Neurons
Mechanotransduction, the conversion of a mechanical stimulus into a biological response, constitutes the basis of a variety of physiological functions such as the senses of touch, balance, proprioception, blood pressure, and hearing. In vertebrates, mechanosensation is mediated by mechanosensory neurons, whose cell bodies are located in trigeminal and dorsal root ganglia. Here, we describe an in vitro model of mechanotransduction that provides an opportunity to explore the properties of mechanosensitive channels in mammalian sensory neurons. The mechano-clamp method allows applying local force on plasma membrane of whole-c...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Perforated Whole-Cell Patch-Clamp Recording
Perforated whole-cell patch-clamp is a variant of the patch-clamp technique used to measure the sum activity of ion channels in the plasma membrane of a single cell. Its defining feature is that electrical access to the cell is obtained through inclusion of a pore-forming antibiotic in the patch pipette which perforates the sealed patch of membrane in contact with the patch pipette. The antibiotic pores allow equilibration of small monovalent ions between the patch pipette and the cytosol whilst maintaining endogenous levels of divalent ions such as Ca2+ and signalling molecules such as cAMP. Therefore, the perforated patc...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Combined Single-Channel and Macroscopic Recording Techniques to Analyze Gating Mechanisms of the Large Conductance Ca2+ and Voltage Activated (BK) Potassium Channel
Ion channels are integral membrane proteins that regulate membrane potentials and signaling of cells in response to various stimuli. The patch-clamp technique enables the study of single channels or a population of channels. The macroscopic recording approaches are powerful in revealing population-averaged behaviors of channels both under basal conditions and in response to various stimuli, modulators and drugs. On their own, however, these approaches can be insufficient for determinations of channel gating mechanisms as they do not accurately report channel open probabilities below 10−2 to 10−3. This obstacle ...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Viral Gene Delivery: Optimized Protocol for Production of High Titer Lentiviral Vectors
HIV-derived lentiviral vectors (LVV) are among the most commonly used gene delivery vehicles. Their production in high quantities, which enables concentration of viral particles to high titers, is important for their successful application in both biomedical research and gene therapy. LVV are produced by co-transfection of three or more plasmids into a packaging cell line followed by several purification and concentration steps. Protocols currently in circulation differ from each other but the direct comparison of their efficacy based on the published information is extremely difficult because more than one variable may be...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Transient Overexpression of Genes in Neurons Using Nucleofection
Nucleofection is a transfection method used to introduce substrates such as cDNA plasmids into primary cells or other cell lines. The method can be successfully applied to cells that are considered difficult to transfect or suffer from low transfection efficiency as seen with traditional transfection techniques. Neurons in primary cultures retain many properties of their in vivo state and therefore, in many instances, are considered better experimental systems than immortalized cell lines, thus becoming increasingly desirable cell types for biomedical research. However, being post-mitotic, primary neuronal cultures are par...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Use of Escherichia coli for the Production and Purification of Membrane Proteins
Individual types of ion channels and other membrane proteins are typically expressed only at low levels in their native membranes, rendering their isolation by conventional purification techniques difficult. The heterologous over-expression of such proteins is therefore usually a prerequisite for their purification in amounts suitable for structural and for many functional investigations. The most straightforward expression host, suitable for prokaryote membrane proteins and some proteins from eukaryotes, is the bacterium Escherichia coli. Here we describe the use of this expression system for production of functionally ac...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
