Me gustaría dar las gracias al gran David Carlson, profesor de Inglés en Matsumoto Dental University, por su apoyo en especie al escribir este informe. También me gustaría agradecer a Tadafumi Adachi, un seminarista en nuestro laboratorio, para su cumplimiento como un sujeto en esta investigación.

1. Preparación de los electrodos de EMG <ol><li> Comience por la preparación de nueve electrodos. Ocho de ellos se utilizan para la grabación de las señales de los sitios sobre el músculo, y se trata de un electrodo de referencia. </li><li> Después de conectar los electrodos de registro y referencia para el amplificador, llenar los discos de pasta de electricidad de la conductancia. Cada tipo está bien, pero un tipo de fluido bajo es mejor. </li><li> Antes de colocar los electrodos en la piel, que el sujeto con firmeza, y determinar la ubicación aproximada del músculo masetero en la cara. </li><li> El tendón superior del músculo masetero se une al arco cigomático, y su tendón inferior al ángulo de la mandíbula. </li><li> Coloque los electrodos de registro ocho en un pedazo largo de cinta adhesiva en la igualdad entre los electrodos a distancia, y adjuntar la matriz a la superficie de la piel. El electrodo de registro primero se coloca en el arco cigomático, y el último en el ángulo de la mandíbula. </li><li> Por último, conecte el electrodo de referencia en la punta de la nariz. </li></ol> 2. EMG de grabación <ol><li> Para empezar a grabar, en primer lugar establecer los parámetros del amplificador analógico de varios canales (MEG6100). Ajuste la ganancia de x 500, el filtro de paso bajo a 0,5 Hz, y el filtro de paso alto y 10 kHz. </li><li> Conecte las ocho salidas del amplificador analógico de un convertidor analógico-digital (PCI-MIO-16E-4). La señal digital se procesa con el software LabVIEW con una frecuencia de muestreo de 20kHz. </li><li> Cuando todo está conectado, EMG registro rápido en la goma de mascar apretando en el lado ipsilateral. </li><li> Las huellas de los ocho canales se presentan en la pantalla LabView inmediatamente después del proceso de grabación se ha completado. </li></ol> 3. EPP extracción y la observación de sus características <ol><li> Para extraer el componente del PPE, en primer lugar eliminar el componente de potencial de acción de la EMG en bruto con un corte alto filtro digital (filtro Butterworth). </li><li> Comenzar a aplicar el filtro con una frecuencia de corte de 10 Hz. Una de ondas lentas que se desvía en la dirección positiva o negativa debe aparecer. Esta onda se corresponde con el EPP, y la diferencia en la polaridad refleja la relación sumidero-fuente de la corriente de la placa final. </li><li> Al grabar desde el músculo masetero, la desviación más negativos de la ola que se espera en la traza del sitio de grabación más inferior, y la polaridad debe cambiar alrededor de 3 sitios fuera de ese sitio. </li><li> La magnitud de la componente del PPE se puede medir como un punto máximo de la desviación o un área en la desviación. </li><li> El cambio de polaridad de los componentes del PPE puede ser determinada por el trazado de la onda del PPE de cada canal en contra de un canal de referencia que muestra la desviación más negativo. </li><li> Ahora, por el aumento de la frecuencia de corte, la onda lenta se volverá oscilatorio. La oscilación se incrementará con el aumento de la frecuencia de corte y, en general se hará evidente con una frecuencia de corte de 30 Hz. </li><li> La fase de la onda se invertirá a través de la traza en la que la polaridad de la onda de 10Hz filtrada PPE mostró un retroceso. </li><li> El cambio de fase de la oscilación se puede comprobar con los mismos gráficos que se han aplicado a la onda de 10Hz-filtrada EPP. </li><li> Desde el intervalo entre los vecinos picos positivos y / o negativos de la oscilación, podemos obtener una aproximación de la frecuencia de la oscilación </li></ol> 4. EMG registrar los resultados <img src="/files/ftp_upload/1653/1653Fig1a.jpg" alt="Figura 1 a" /> Figura 1a: Aquí se muestra un esquema de los ocho lugares a lo largo del músculo masetero que se registraron a partir de. <img src="/files/ftp_upload/1653/1653Fig1b.jpg" alt="Figura 1 b" /> Figura 1b: Vemos una muestra de la EMG monopolar registradas simultáneamente en los sitios sobre el músculo masetero, en respuesta a apretar las encías rápido en el lado ipsilateral. El componente de EPP fue extraído por medio de un filtro digital de corte alto con una frecuencia de corte de 10 Hz, que se superpone a cada prima EMG. En este ensayo, la desviación más negativa se observó en los rastros-5 y -6 y la polaridad cambiada a través de huella-3. <img src="/files/ftp_upload/1653/1653Fig1c.jpg" alt="Figura 1c" /> Figura 1c: Cuando la frecuencia de corte del filtro de corte alto digitales se fijó a 30 Hz, la onda lenta exhiben una oscilación. Su fase también se desplazó a través de traza 3, donde la polaridad de la onda de 10Hz-filtrada EPP cambiado. El intervalo entre vecinos picos positivos de la oscilación se midió. En este tema, el promedio entre el intervalo máximo fue de 31,7 ± 6,5 ms más de 16 grabaciones.

<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> A medida que el potencial negativo se considera que es formada por un flujo de de la placa terminal actual, y la desviación positiva, por su caudal de salida [1,2,3,4], la traza que muestra la desviación más negativo debe corresponder a la sitio de localización de la unión neuromuscular [5]. La figura 1b indica la unión neuromuscular del músculo masetero localizar en su parte inferior cerca del ángulo de la mandíbula, que es aproximadamente igual a los resultados obtenidos por otro método que utiliza la forma de conducción d...

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<td>Analogue filter</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>MEG6100</td>
<td>pre-,main-amplifier</td>
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<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>
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<td>Connection interface</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>BNC-2090</td>
<td>8ch BNC adaptor</td>
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<tr>
<td>Disc electrodes</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>NS-11</td>
<td>Ag/AgCl (&Phi;8mm)</td>
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<td>Electrode past</td>
<td>&nbsp;</td>
<td>Sanshin</td>
<td>SA-5</td>
<td>Semi-fluid carbon past</td>
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<td>LabVIEW (Digital filter)</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>V. 8.5</td>
<td>Programming language</td>
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<td>Chewing gum</td>
<td>&nbsp;</td>
<td>Lotte</td>
<td>Green gum</td>
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extraction of the epp component from the surface emg

Un electromiograma de superficie (EMG), especialmente cuando se registraron cerca de la unión neuromuscular, se espera que contenga el potencial de placa terminal (EPP), componente que se puede extraer con un filtro de señal adecuada. Hay dos factores importantes: el Grupo de Gestión Ambiental debe ser registrada en forma monopolar, y la grabación se debe hacer para que la señal de baja frecuencia que corresponde el PPE no se elimina. Este informe explica cómo extraer el componente PPE de la EMG del músculo masetero en un ser humano. La superficie EMG se registran de ocho sitios con electrodos de disco tradicionales alineados a lo largo sobre el músculo, con la misma distancia entre los electrodos del arco cigomático hasta el ángulo de la mandíbula en respuesta a apretar la goma rápida. Un electrodo de referencia se coloca en la punta de la nariz. El componente de EPP se extrae de la EMG cruda mediante la aplicación de una alta corte de filtro digital (segunda dimensión filtro Butterworth) con un rango de 10-35 Hz. Cuando el filtro se establece en 10 Hz, los extraídos del PPE ola desvía positiva o negativa dependiendo del lugar de grabación. La diferencia en la polaridad refleja la relación sumidero-fuente de la corriente de placa final, con el sitio que muestra la desviación más negativa corresponde a la unión neuromuscular. En el caso del músculo masetero, la unión neuromuscular se estima que se ubicará en la parte inferior cerca del ángulo de la mandíbula. El componente del PPE presenta una oscilación interesante cuando la frecuencia de corte del filtro de corte alto digitales es de 30 Hz. La oscilación del PPE indica que la contracción muscular se ajusta de manera intermitente. Temblores anormales que acompañan a varios tipos de enfermedades pueden ser sustancialmente debido a la oscilación del PPE, que se vuelve más lento y es difícil de dejar.

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

El potencial de placa terminal (EPP) componente se puede extraer de la superficie EMG utilizando un filtro digital. La muestra extraída del PPE oscilación con una frecuencia de alrededor de 30 Hz.

La extracción de los componentes del PPE de la EMG de superficie

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 vistas.  Matsumoto Dental University. El potencial de placa terminal (EPP) componente se puede extraer de la superficie EMG utilizando un filtro digital. La muestra extraída del PPE oscilación con una frecuencia de alrededor de 30 Hz.

Video: 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-disección de cerebro de rata para el ARN o la extracción de proteínas a partir de región específica del cerebro

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

Investigación

Biología

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

La extracción de RNA a partir de células Neuroprecursor utilizando el kit de Bio-Rad ARN total

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.

Etiquetado retrógrada de las células ganglionares de la retina por la aplicación de Fluoro-Gold en la superficie del colículo superior

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

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificación de los progenitores del músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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.

Avidez detección basada en la interacción extracelular (AVEXIS) para la detección escalable de extracelular de baja afinidad interacciones ligando-receptor

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.

Florecimiento extracción de proteínas de levadura y purificación para el Estudio de la función, las interacciones y modificaciones post-traduccionales

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.

Mediciones en tiempo real de proteínas de membrana: Receptor interacciones utilizando resonancia de plasmones superficiales (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.

La técnica de infiltración-centrifugación para extracción de apoplástico fluido procedente de hojas de la planta Uso<em&gt; Phaseolus vulgaris</em&gt; Como ejemplo

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.

Imágenes en tiempo real de la Planta Dinámica superficie celular con Variable de ángulo epifluorescencia Microscopía

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...