Ich möchte großen Dank an David Carlson, Professor für Englisch an der Matsumoto Dental University, bieten für die freundliche Unterstützung beim Schreiben dieses Berichts. Ich möchte auch Tadafumi Adachi, ein Seminarist in unserem Labor für seine Compliance als Thema in dieser Untersuchung danken.

1. Vorbereitung der EMG-Elektroden <ol><li> Beginnen Sie mit der Vorbereitung neun Elektroden. Acht von ihnen sind für die Aufnahme Signale von Websites, auf den Muskel eingesetzt, und man ist eine Referenz-Elektrode. </li><li> Nach dem Anschließen des Aufzeichnungs-und Referenz-Elektroden an den Verstärker, füllen Sie die Scheiben mit elektrischen Leitwert einfügen. Jeder Typ ist OK, aber eine niedrige Fluid ist besser. </li><li> Vor dem Anbringen der Elektroden auf die Haut, haben das Thema fest nach unten, und bestimmen Sie die ungefähre Lage des M. masseter auf dem Gesicht. </li><li> Die überlegene Sehne des M. masseter wird an der Jochbogen und seine untere Sehne um den Winkel des Unterkiefers. </li><li> Legen Sie die acht Aufnahmen Elektroden auf ein langes Stück Klebeband in gleich zwischen den Elektroden entfernt, und befestigen Sie das Array auf der Hautoberfläche. Die erste Aufnahme Elektrode auf der Jochbogen und der letzte auf den Winkel des Unterkiefers gelegt. </li><li> Schließlich legen die Bezugselektrode auf die Nasenspitze. </li></ol> 2. EMG-Aufzeichnung <ol><li> Aufnahme zu beginnen, legen Sie zunächst die Parameter der Multi-Kanal-Analog-Verstärker (MEG6100). Stellen Sie die Verstärkung auf x 500, der Tiefpassfilter auf 0,5 Hz, und die Hochpassfilter bis 10 kHz. </li><li> Schließen Sie die acht Ausgänge der analogen Verstärker zu einem Analog-Digital-Wandler (PCI-MIO-16E-4). Das digitale Signal wird mit LabView-Software mit einer Abtastrate von 20 kHz verarbeitet. </li><li> Wenn alles angeschlossen ist, aufnehmen EMGs bei schnellen Kaugummi ballt auf der ipsilateralen Seite. </li><li> Die Spuren aus den acht Kanäle sind auf der LabView-Display sofort nach der Aufnahme abgeschlossen ist vorgestellt. </li></ol> 3. EVP-Extraktion und Beobachtung ihrer Funktionen <ol><li> Zur Extraktion der EVP-Komponente, entfernen Sie zunächst das Aktionspotential Komponente aus dem Roh-EMG mit Hilfe eines High-Cut digitale Filter (Butterworth-Filter). </li><li> Beginnen Sie die Anwendung der Filter mit einer Grenzfrequenz von 10 Hz. Eine langsame Welle, die in die positive oder negative Richtung lenkt erscheinen soll. Diese Welle entspricht der EVP, und der Unterschied in der Polarität spiegelt die sink-source Verhältnis der Endplatte Strom. </li><li> Bei der Aufnahme von M. masseter, ist die negative Auslenkung der Welle in die Spur aus den minderwertigen Aufnahmen vor Ort erwartet, und die Polarität sollte ca. 3 Seiten weg von dieser Site. </li><li> Die Größe der EVP-Komponente kann als Gipfelpunkt der Ablenkung oder eine Fläche bei der Auslenkung gemessen werden. </li><li> Die Änderung der Polarität des EVP-Komponente kann durch Auftragen der EVP-Welle von jedem Kanal gegen eine Referenz-Kanal, der die negative Auslenkung zeigte ermittelt werden. </li><li> Jetzt durch die Erhöhung der Grenzfrequenz, wird die langsame Welle geworden oszillatorische. Die Schwingung wird mit zunehmender cut-off Frequenz zu erhöhen und wird in der Regel werden bei einem Cut-off Frequenz von 30 Hz auf der Hand. </li><li> Die Phase der Welle wird über die Spur, in der die Polarität der 10Hz gefiltert EVP-Welle zeigte eine Umkehr umkehren. </li><li> Die Phasenverschiebung der Schwingung kann mit der gleichen Grafiken, die auf die 10Hz-gefiltert EVP Welle aufgebracht wurden überprüft werden. </li><li> Aus dem Intervall zwischen benachbarten positive und / oder negative Spitzen der Schwingung, können wir eine Annäherung an die Frequenz der Schwingung </li></ol> 4. EMG-Aufzeichnung Ergebnisse <img src="/files/ftp_upload/1653/1653Fig1a.jpg" alt="Abbildung 1 a" /> Abbildung 1a: Dargestellt ist hier eine schematische Darstellung der acht Standorten entlang des M. masseter, die aus aufgenommen wurden. <img src="/files/ftp_upload/1653/1653Fig1b.jpg" alt="Abbildung 1 b" /> Abbildung 1b: Wir sehen eine Probe der monopolaren EMGs gleichzeitig von den Seiten über den Masseter in Reaktion auf schnelle gum Pressen auf der ipsilateralen Seite aufgezeichnet. Die EVP-Komponente extrahiert wurde mit Hilfe eines High-Cut digitales Filter mit einer Grenzfrequenz von 10 Hz, die auf jeder EMG überlagert wird. In dieser Studie war die negative Ausschlag in Spuren-5 und -6 beobachtet und die Polarität zwischen Spur-3 geändert. <img src="/files/ftp_upload/1653/1653Fig1c.jpg" alt="Abbildung 1c" /> Abbildung 1c: Wenn die Cut-Off-Frequenz des high-cut digitale Filter 30Hz eingestellt wurde, zeigte die langsame Welle eine Schwingung. Seine Phase auch über Spur 3, wobei die Polarität der 10Hz-gefiltert EVP Welle verändert verschoben. Das Intervall zwischen den benachbarten positiven Spitzen der Schwingung gemessen wurde. In diesem Thema war die durchschnittliche Inter-Peak-Intervall 31,7 ± 6,5 msec über 16 Aufnahmen.

<ol>
	<li>Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physiology of synapses. , (1964).</li><li>Mitzdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+source-density+method+and+application+in+cat+cerebral+cortex:+investigation+of+evoked+potentials+and+EEG+phenomena.">Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena.</a> Physiol. Rev. 65, 37-100 (1985).</li><li>Rall, W., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+reconstruction+of+field+potentials+and+dendrodendritic+synaptic+interactions+in+olfactory+bulb.">Theoretical reconstruction of field potentials and dendrodendritic synaptic interactions in olfactory bulb.</a> J. Neurophysiol. 31, 884-915 (1968).</li><li>Richardson, T. L., Turner, R. W., Miller, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action-potential+discharge+in+hippocampal+CA1+pyramidal+neurons:+current+source+density+analysis.">Action-potential discharge in hippocampal CA1 pyramidal neurons: current source density analysis.</a> J. Neurophysiol. 58, 981-996 (1987).</li><li>Kumai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+the+neuromuscular+junction+of+the+human+masseter+muscle+estimated+from+the+low+frequency+component+of+the+surface+electromyogram.">Location of the neuromuscular junction of the human masseter muscle estimated from the low frequency component of the surface electromyogram.</a> J. Jpn. Physiol. 55, 61-68 (2005).</li><li>Mito, K., Sakamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+muscle+fiber+conduction+velocity+of+m.+masseter+during+voluntary+isometric+contraction.">Distribution of muscle fiber conduction velocity of m. masseter during voluntary isometric contraction.</a> Electroencephalogr. Clin. Neurophysiol. 40, 275-285 (2000).</li><li>Tokunaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+configuration+of+the+myoneural+junctions+of+human+masticatory+muscle+detected+with+matrix+electrode.">Two-dimensional configuration of the myoneural junctions of human masticatory muscle detected with matrix electrode.</a> J. Oral Rehabili. 25, 329-334 (1998).</li><li>Rohen, J. H., Yokochi, C., L&#x0171;tjen-Drecoll, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Color atlas of anatomy. , (2006).</li><li>Wichmann, T., DeLong, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillation+in+the+basal+ganglia.">Oscillation in the basal ganglia.</a> Nature. 400, 621-622 (1999).</li></ol>

<ol><li> Als negatives Potential gilt als durch eine Strömung in der Endplatte Strom, und die positiven Ausschlag gebildet werden, durch die flow-out [1,2,3,4], muss die Spur, welche die meisten negativen Ausschlag nach entsprechen site Ortung der neuromuskulären Synapse [5]. Abbildung 1b zeigt der neuromuskulären Synapse des M. masseter in seinem unteren Teil in der Nähe zu finden, um den Winkel des Unterkiefers, die ungefähr die gleiche ist wie Ergebnisse mit einer anderen Methode unter Verwendung der Leitung Weise der Motoreinheit Aktio...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Analogue filter</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>MEG6100</td>
<td>pre-,main-amplifier</td>
</tr>
<tr>
<td>A/D converter</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-MIO-16E-4</td>
<td>PCI board for computer</td>
</tr>
<tr>
<td>Connection interface</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>BNC-2090</td>
<td>8ch BNC adaptor</td>
</tr>
<tr>
<td>Disc electrodes</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>NS-11</td>
<td>Ag/AgCl (&Phi;8mm)</td>
</tr>
<tr>
<td>Electrode past</td>
<td>&nbsp;</td>
<td>Sanshin</td>
<td>SA-5</td>
<td>Semi-fluid carbon past</td>
</tr>
<tr>
<td>LabVIEW (Digital filter)</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>V. 8.5</td>
<td>Programming language</td>
</tr>
<tr>
<td>Chewing gum</td>
<td>&nbsp;</td>
<td>Lotte</td>
<td>Green gum</td>
<td>Test food in clenching</td>
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extraction of the epp component from the surface emg

Eine Oberfläche Elektromyogramm (EMG), vor allem, wenn in der Nähe der neuromuskulären Synapse aufgenommen, wird erwartet, dass die Endplatte Potential (EPP)-Komponente, die mit einem entsprechenden Signal-Filter extrahiert werden kann, enthalten. Zwei Faktoren sind wichtig: Die EMG ist in monopolaren Mode aufgezeichnet werden, und die Aufnahme getan werden muss, so die tiefen Frequenzen entsprechend der EVP nicht beseitigt ist. Dieser Bericht erläutert, wie die EVP-Komponente aus dem EMG des M. masseter Extrakt in einem menschlichen Subjekt. Die Oberflächen-EMG wird von acht Standorten mit traditionellen Scheiben-Elektroden entlang über den Muskel ausgerichtet aufgezeichnet, mit der gleichen inter-Elektrode Abstand von der Jochbogen, um den Winkel des Unterkiefers in Antwort auf schnelle gum ballte. Eine Referenzelektrode ist an der Spitze der Nase platziert. Die EVP-Komponente aus dem Roh-EMG durch Anlegen eines High-Cut digitale Filter (2. Dimension Butterworth-Filter) mit einer Reichweite von 10-35 Hz extrahiert. Wenn der Filter auf 10 Hz eingestellt ist, lenkt die extrahierten EVP-Welle entweder negativ oder positiv abhängig von der Aufnahme vor Ort. Der Unterschied in der Polarität spiegelt die sink-source Verhältnis der Endplatte Strom, mit dem Standort zeigt die negative Auslenkung entspricht der neuromuskulären Synapse. Im Falle des M. masseter ist der neuromuskulären Synapse schätzungsweise in den unteren Teil nahe dem Winkel des Unterkiefers befinden. Die EVP-Komponente stellt eine interessante Schwingung, wenn der Cut-Off-Frequenz des high-cut digitale Filter mit 30 Hz eingestellt ist. Die EVP Schwingung bedeutet, dass die Muskelkontraktion ist in einer intermittierenden Weise eingestellt. Abnormal Zittern begleitet verschiedene Arten von Krankheiten können im Wesentlichen aufgrund dieser EPP Schwingung, die langsamer wird und ist schwer zu hören.

Watch this Scientific Journal Video about Extraction of the EPP Component from the Surface EMG at JoVE.com

Extraction of the EPP Component from the Surface EMG

Die Endplatte Potential (EPP)-Komponente kann von der Oberfläche EMG extrahiert werden mit einem digitalen Filter. Die extrahierten EVP zeigt Schwingung mit einer Frequenz von etwa 30 Hz.

Extraction der EVP-Komponente aus dem Oberflächen-EMG

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component ...

extraction-of-the-epp-component-from-the-surface-emg

Research

JoVE Journal

Biology

12.5K Aufrufe.  Matsumoto Dental University. Die Endplatte Potential (EPP)-Komponente kann von der Oberfläche EMG extrahiert werden mit einem digitalen Filter. Die extrahierten EVP zeigt Schwingung mit einer Frequenz von etwa 30 Hz.

Serie: Extraction of the EPP Component from the Surface EMG

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-Dissektion Rattenhirns RNA oder Protein Extraction von bestimmten Hirnregion

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Forschung

Biologie

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

RNA Extraktion aus Neuroprecursor Zellen unter Verwendung des Bio-Rad Total RNA Kit

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.

This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.

Retrograde Markierung von retinalen Ganglienzellen durch Anwendung von Fluoro-Gold auf der Oberfläche des Colliculus superior

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidität-basierte extrazelluläre Wechselwirkung Screening (AVEXIS) für die Erkennung von Scalable Low-Affinität extrazellulären Rezeptor-Ligand-Wechselwirkungen

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bäckerhefe Protein Extraktion und Reinigung für das Studium der Funktion, Wechselwirkungen, und post-translationale Modifikationen

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Echtzeit-Messungen von Membranprotein: Rezeptor-Wechselwirkungen Mit Oberflächenplasmonenresonanz (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

Die Infiltration-Zentrifugation Technik zur Gewinnung von apoplastischen Flüssigkeit aus Pflanzenblättern verwenden<em&gt; Phaseolus vulgaris</em&gt; Als Beispiel

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

Echtzeit-Imaging von Plant Cell Oberflächen Dynamics mit variabler Winkel Epifluoreszenzmikroskopie

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Methoden zur Extraktion von Endosymbionten aus der Whitefly<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

In Vivo Surface Elektrokardiographie für erwachsene Zebrafische

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...