1. Preparación de soluciones y materiales Estos pasos deben llevarse a cabo de varios días de antelación. <ol><li> Cubreobjetos de vidrio. Cubreobjetos fueron limpiados por inmersión durante 10 minutos en una solución de detergente Linbro 7X: agua, mezclado en una relación 1: 3 relación y se calienta con agitación hasta que se aclare. Cubreobjetos se lavaron exhaustivamente con agua destilada, y luego se hornea a 450 ° C durante 6 horas. Cargando cubreobjetos en bastidores tinción de cerámica (ver reactivos) simplifica este proceso. </li><li> Proteína solución para la estampación. Reconstituir la fibronectina siguiendo las instrucciones de los fabricantes una solución madre de 1 mg / ml de concentración. </li></ol> 2. Fundido sellos del maestro topológica Estos pasos pueden llevarse a cabo de varios días de antelación. Sellos tienda de patrones en un plato cubierto, como una placa de cultivo de tejidos. <ol><li> Quite el polvo del maestro por medio de una corriente de aire comprimido, aire filtrado o gas inerte. </li><li> Coloque el maestro, con dibujos laterales arriba, en el fondo de un plato de plástico apenas más grande que el maestro. 60 - o 100 mm de placas de cultivo de tejidos son muy adecuadas para este propósito. </li><li> En un tubo de poliestireno de centrífuga de 50 ml, se combinan los componentes Sylgard en una proporción de agente de curado: base de elastómero de 1: 10 en peso. Mezcle bien con un dispositivo de plástico, como una pipeta desechable. Preparar al menos 0,2 ml de elastómero por centímetro cuadrado de área de plato. </li><li> Centrifugar el tubo de 50 ml a 300 g durante 5 min para eliminar las burbujas de aire. </li><li> Vierta el elastómero sobre el maestro, y luego colocarlo en un secador, al vacío, durante 30 minutos. </li><li> Curar el elastómero en un horno a 65 ° C durante al menos 2 horas. Curado a temperaturas más altas y durante más tiempo los resultados en tiempos más rígido elastómero. Deje que se enfríe a temperatura ambiente. </li><li> Separar la hoja de sellos del maestro. </li></ol> 3. Microcontacto de impresión de la fibronectina sobre vidrio <ol><li> Corte un único sello. Sellos de medición de 4 mm x 4 mm a 1 cm x 1 cm en la zona y 1 a 2 mm de espesor son más fáciles de empezar. Ponga el lado patrón de arriba en un portaobjetos de cristal o de plástico. </li><li> Coloque el sello en un limpiador de plasma, y ​​el proceso, al vacío, durante 30 segundos. Un limpiador de plasma Harrick Científico (ver equipos), establece en su valor más alto de salida hará que la superficie hidrofílica PDMS. Ya veces provocar el agrietamiento del elastómero. </li><li> Diluir la solución de fibronectina con agua desionizada a una concentración de estampación de 50 ug / ml. </li><li> Coloque una pequeña gota (10 - 50 l) de solución de estampado en el sello. Se extenderá por todo la superficie hidrófila. Agregar suficiente solución única que cubre la caída de la marca, pero no se ejecuta en los bordes. Vamos a absorber las proteínas para acabar por 5 minutos. </li><li> El uso de un Kimwipe ® u otro tejido de papel limpia, la mecha de la mayor parte de la solución de proteína de la marca, sin tocar la región con patrón. </li><li> Seque el resto de la solución de la marca bajo un chorro de agua limpia, el gas seco e inerte como el nitrógeno. </li><li> Con unas pinzas, retire el sello de la lámina de vidrio, invertir, y el lugar en contacto con el cubreobjetos limpio (la superficie a rellenar con patrón). Colocar un peso encima para promover un buen contacto. El peso específico que proporciona el mejor patrón depende del tamaño de la estampilla y el patrón, empieza con un peso de 5 g, y ajuste entre piezas estampadas. Deje el sello en contacto con la superficie de un minuto. </li><li> Con cuidado, desmontar la pila, y el sello independiente de cubreobjetos. </li><li> Enjuague vigorosamente el cubreobjetos con dibujos en PBS, seguido de agua destilada, para eliminar la proteína que no se adsorbe a la superficie. Cubreobjetos seca en corriente de nitrógeno. </li></ol> Resultados representante Impresión por microcontacto es un proceso de gran alcance para las moléculas de los patrones en las superficies. Este proceso tiene la capacidad de crear funciones con dimensiones que van desde decenas de micrómetros a cientos de nanómetros, en la figura. 1A, los logotipos de la izquierda son cada 200 m de altura, mientras que las manchas verdes se ilustra en la figura. 1B son: 1 m de diámetro y espaciados a intervalos de 4 m, medidos de centro a centro. Fig. 1B también ilustra una propiedad de gran alcance de impresión por microcontacto, es decir, que es y el proceso aditivo. Múltiples rondas de impresión por microcontacto se puede aplicar a una sola superficie para crear sistemas multicomponentes 3. <img src="/files/ftp_upload/old/1065_2008_11_24_3/Figure_a.jpg" alt="Figura A" /><img src="/files/ftp_upload/old/1065_2008_11_24_4/FIGURE_B.jpg" alt="Figura B" />

<ol>
	<li>Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cell+life+and+death.">Geometric control of cell life and death.</a> Science. 276, 1425-1425 (1997).</li><li>Kumar, A., Whitesides, 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=Features+of+Gold+Having+Micrometer+to+Centimeter+Dimensions+can+be+Formed+Through+a+Combination+of+Stamping+with+an+Elastomeric+Stamp+and+an+Alkanethiol+"Ink"+Followed+by+Chemical+Etching.">Features of Gold Having Micrometer to Centimeter Dimensions can be Formed Through a Combination of Stamping with an Elastomeric Stamp and an Alkanethiol "Ink" Followed by Chemical Etching.</a> Applied Physics Letters. 63, 4-4 (1993).</li><li>St. John, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+Glial+Cell+Attachment+to+Microcontact-printed+Surfaces.">Preferential Glial Cell Attachment to Microcontact-printed Surfaces.</a> Journal of Neuroscience Methods. 75, 171-171 (1997).</li><li>Kam, L., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+to+protein-micropatterned-supported+lipid+bilayer+membranes.">Cell adhesion to protein-micropatterned-supported lipid bilayer membranes.</a> Journal of Biomedical Materials Research. 55, 487-487 (2001).</li><li>Kung, L. A., Kam, L., Hovis, J. S., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Hybrid+Surfaces+of+Proteins+and+Supported+Lipid+Bilayers.">Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers.</a> Langmuir. 16, 6773-6773 (2000).</li><li>Shen, K., Thomas, V. K., Dustin, M. L., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+of+costimulatory+ligands+enhances+CD4++T+cell+function.">Micropatterning of costimulatory ligands enhances CD4+ T cell function.</a> Proceedings of the National Academy of Sciences. 105, 7791-7791 (2008).</li><li>Shi, P., Shen, K., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+presentation+of+L1+and+N-cadherin+in+multicomponent,+microscale+patterns+differentially+direct+neuron+function+in+vitro.">Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro.</a> Developmental Neurobiology. 67, 1765-1765 (2007).</li></ol>

The authors have nothing to disclose.

El proceso de impresión por microcontacto es conceptualmente simple y muy robusto, que se ha aplicado a los patrones de una amplia gama de moléculas en una variedad de sustratos. Sin embargo, este proceso sigue siendo algo de un arte. La geometría específica del modelo que se creó, la proteína a ser modelado, el peso aplicado, y el recubrimiento / sellado todas las condiciones de afectar la calidad de estampado. Por ejemplo, poco peso, aplicada a grandes rasgos, a menudo resulta en diferencias en el patrón como se puede ver en el log...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Plasma Cleaner</td>
<td>&nbsp;</td>
<td>Harrick Scientific Products, Inc.</td>
<td>PDC-32G</td>
<td>&nbsp;</td>
<tr>
<td>Desiccator</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5315-0150</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10010-072</td>
<td>&nbsp;</td>
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<td>Protein labeling kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A30006</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin </td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F2006</td>
<td>&nbsp;</td>
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<td>Staining rack</td>
<td>Reagent</td>
<td>Thomas Scientific</td>
<td>8542E40</td>
<td>&nbsp;</td>
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<td>Coverslips</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>12-544-12</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>Reagent</td>
<td>Ellsworth Adhesives</td>
<td>184 Sil Elast Kit</td>
<td>&nbsp;</td>
<tr>
<td>Diffraction Grating</td>
<td>Reagent</td>
<td>Edmund Scientific</td>
<td>3040267</td>
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microcontact printing of proteins for cell biology

La capacidad de las proteínas y otras biomoléculas patrón sobre sustratos es importante para captar la complejidad espacial del medio ambiente extracelular. Desarrollo de la imprenta microcontacto por el grupo de Whitesides (<a href="http://gmwgroup.harvard.edu/"&gt; Http://gmwgroup.harvard.edu/</a&gt;) A mediados de la década de 1990 revolucionó este campo, haciendo de la microelectrónica / técnicas de microfabricación acceso a los laboratorios se centraron en las ciencias de la vida. Las implementaciones iniciales de este método de polidimetilsiloxano (PDMS) sellos para crear patrones de productos químicos en las superficies de materiales funcionalizados<sup&gt; 1</sup&gt;. Desde entonces, una serie de enfoques innovadores han sido desarrollados para las moléculas de otro patrón, incluyendo las proteínas<sup&gt; 2</sup&gt;. Este vídeo muestra el proceso básico de creación de sellos de PDMS y las utiliza para las proteínas patrón, ya que estas medidas son difíciles de expresar con precisión en las palabras. Nos centramos en los patrones de la matriz proteica extracelular fibronectina en cubreobjetos de vidrio como un ejemplo específico de los patrones. Un componente importante del proceso de impresión por microcontacto es un maestro topológica, de la que los sellos son emitidos, las regiones de subir y bajar del maestro se reflejan en la marca y definir el modelo final. Por lo general, un maestro consiste en una oblea de silicio recubierto con fotorresistencia y modelada por fotolitografía como se hace aquí. La creación de los maestros que contienen un patrón específico requiere equipo especializado, y se aborda mejor en consulta con un centro de fabricación o instalación. Sin embargo, casi cualquier sustrato con la topología se puede utilizar como un maestro, como redes de difracción de plástico (ver Reactivos para un ejemplo), y como maestros casual proporcionar fácilmente disponible, los patrones de simple. Este protocolo se inicia en el punto de tener un maestro en la mano.

Watch this Scientific Journal Video about Microcontact Printing of Proteins for Cell Biology at JoVE.com

Microcontact Printing of Proteins for Cell Biology

Impresión por microcontacto se utiliza ampliamente a las proteínas de patrones y otras moléculas en las superficies de material. Se demuestra los pasos básicos de este proceso, los patrones de estampación de la fibronectina sobre el cristal.

Microcontacto de impresión de las proteínas de Biología Celular

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

microcontact-printing-of-proteins-for-cell-biology

Research

JoVE Journal

Biology

19.9K vistas.  Columbia University. Impresión por microcontacto se utiliza ampliamente a las proteínas de patrones y otras moléculas en las superficies de material. Se demuestra los pasos básicos de este proceso, los patrones de estampación de la fibronectina sobre el cristal.

Video: Microcontact Printing of Proteins for Cell Biology

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un dispositivo de microfluidos gradiente de generación de Biología Celular

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Investigación

Biología

Micro-scale Engineering for Cell Biology

Micro-escala de Ingeniería de Biología Celular

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Etiquetado selectiva de las proteínas de la superficie celular con CyDye DIGE Colorantes Fluor mínimo

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

La evaluación de dos dimensiones ensayos de cristalización de proteínas de membrana pequeña de Estudios de Biología Estructural de Cristalografía de electrones

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Marcaje fluorescente de COS-7 Expresando etiqueta SNAP-proteínas de fusión de imágenes de células vivas

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

De proteínas de membrana de superposición de ensayo: Un protocolo de prueba de interacción entre las proteínas soluble e insoluble In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Desplegable de proteínas de unión a calmodulina

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado levadura sistema híbrido de dos para identificar proteínas que interactúan con el factor de crecimiento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

El uso de quimiostatos en Microbiana Biología de Sistemas

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...