Esta investigación fue apoyada por la ISF, NSF, y ERC. Queremos reconocer la ayuda técnica con la etapa de temperaturas de Randy Milford, Michael Koren, Shafer Doug y Jeremy Dennison. Asistencia en el desarrollo de software fue proporcionado por O Chen, Xu Di, Sannareddy Rajesh, y Bhattachary Sumit. Nos gustaría agradecer a nuestros colaboradores el profesor Peter L. Davies y el Dr. Laurie A. Graham para la proteína de fusión IBP y útiles debates. También queremos agradecer a los miembros del laboratorio del Dr. Maya Bar-Dolev, Qin Yangzhong, Dr. Yeliz Celik, el Dr. Natalya Pertaya, Mizrahy Ortal, y Guy Shlomit por su retroalimentación de los usuarios.

0. Procedimientos preliminares <ol><li> Capilar de vidrio para inyección solución. Usando un extractor capilar (Narishige, Tokyo, Japón), preparar una pipeta agudo con una abertura fina de un tubo capilar de vidrio micro (Marca GMBH, Wertheim, Alemania). El tamaño de la abertura debe ser verificada haciendo pasar aire a través del capilar para obtener burbujeo fino en agua limpia. Si el capilar se cierra, entonces se puede abrir rompiendo su borde. Esto puede conseguirse pulsando o rascarse suavemente contra el agua que contiene paredes de los tubos. Preparar el capilar tal que la abertura está casi bloqueado, pero es lo suficientemente abierto para permitir la formación de burbujas de menos de un milímetro. </li><li> Limpieza Cobre disco. Sonicar los discos de cobre durante 10 minutos en 0,1% Micro-90 jabón (Cole-Parmer, Vernon Hills, Illinois, EE.UU.), luego lavar con agua doblemente destilada. Introducir los discos en una mezcla de isopropanol (técnico) y sonicar solución de nuevo durante 10 min. Aletaaliado, se secan los discos con aire filtrado. Esta etapa de limpieza es crítica para evitar la contaminación IBP entre los experimentos. </li><li> Doble capa de montaje cubreobjetos. Un conjunto de cubreobjetos se preparó para permitir la observación de la muestra sin condensación de humedad sobre la superficie de vidrio cubierta. Esto se logró mediante la colocación de un Drierite (WA Hammond Drierite, Xenia, Ohio, EE.UU.) partícula (2 mm de diámetro) entre dos cubreobjetos que fueron pegados a continuación con una pistola de pegamento caliente. Esta configuración impide la condensación que podría bloquear la vista cuando la muestra se enfrió a bajas temperaturas y elimina la necesidad para soplar aire seco a la ventana de observación. </li></ol> 1. Etapa de enfriamiento Set-up <ol><li> Conectar la entrada de flujo de agua y la salida de la etapa de enfriamiento a 4 mm de diámetro interno Tygon tubos (Saint-Gobain, París, Francia), y conecte el flujo de agua del tubo de entrada a una bomba de agua. </li><li> Conectar un tubo de 4 mm interior Tygon diámetro a la entrada de la etapa de enfriamiento to suministrar aire seco. El aire se secó usando una columna en línea de Drierite. </li><li> Operar las bombas de aire y agua. Tenga en cuenta que los elementos de refrigeración no debe funcionar sin un disipador de calor. </li><li> Encienda el controlador de temperatura, cámara, y la rutina de LabVIEW. </li></ol> 2. Preparación de la muestra <ol><li> Colocar una gota de 3-4 l de aceite de inmersión B (Cargille laboratorios, Cedar Grove, Nueva Jersey, EE.UU.) en el lado posterior de un disco de diámetro de cobre de 7 mm que tiene 500 agujeros perforados mu m a través del disco. </li><li> Colocar el disco de cobre en la etapa de enfriamiento con el lado del aceite de inmersión hacia abajo. </li><li> Conectar el tubo capilar (el borde romo) a un 0,7 mm de diámetro interior del tubo Tygon conectado en el otro extremo a una jeringa de 2 ml, de vidrio (Poulten-Graf, Wertheim, Alemania). </li><li> Antes de utilizar el tubo capilar, visita de la pequeña abertura de la capilaridad para asegurar que la abertura es un tamaño apropiado (véase los procedimientos preliminares). </li><li> Inserte lentamente la glass capilar en el tubo de muestra preparada IBP proteína (2,4 mM Mp IBP-GFP en 20 mM de CaCl 2 y 25 mM Tris-HCl a pH 8, véase la referencia 10 para los detalles de preparación) y tirar de la jeringa de vidrio hasta que el capilar de vidrio contiene 0,1 l de la solución de proteína. </li><li> Comienza la grabación de vídeo a través del software LabVIEW. </li><li> Insertar el borde afilado de la capilar de vidrio (que contiene la solución de proteína) en uno de los agujeros en el disco de cobre en la etapa de enfriamiento. </li><li> Mientras observa a través del microscopio (Olympus, Tokio, Japón, objetivo 10x), cuidadosamente penetrar en la capa de inmersión en aceite con la punta capilar de vidrio, y presione la jeringa de vidrio (muy delicadamente) para suministrar una cantidad pequeña (~ 10 nl) de la proteína solución para crear una gotita 200 micras. </li><li> Cubrir el agujero en la etapa de enfriamiento con el conjunto de capa doble cubreobjetos (véase los procedimientos preliminares). </li></ol> 3. TH de medición de actividad <ol><li> Press el botón de refrigeración y la temperatura a -40 ° C. </li><li> Inicialmente, la gota de solución será clara. A temperaturas bajas, típicamente en el rango de -30 ° C a -35 ° C, el color gotita cambios, lo que indica que la solución ha sido congelado. Inmediatamente después de la muestra se ha congelado, aumentar la temperatura lentamente hasta que la mayor parte de hielo comienza a derretirse. Un aumento gradual de la temperatura es necesario para evitar el rebasamiento de la temperatura que podría resultar en la fusión completa de la muestra. </li><li> Cambiar a un objetivo 50x y comienzan a fundir el hielo mediante el ajuste de la temperatura. Este ajuste es interactivo, y los pasos finales se realiza normalmente con pequeños pasos de temperatura de 0,002 ° C. Continúe hasta que se derrita un cristal único que queda. El tamaño final del cristal debe ser alrededor de 10 micras. La temperatura más alta a la que ha dejado de fusión se determina que el punto de fusión y se determina con precisión en la etapa de análisis de vídeo más tarde. </li> &lt;li&gt; Ajustar la temperatura a unas pocas centésimas de un grado centígrado por debajo del punto de fusión del cristal y comenzar una rampa de temperatura con un retraso de 10 min. Ajustar la velocidad de rampa según se desee. Durante este tiempo, el cristal estará expuesto a los IBP. </li><li> Al finalizar el tiempo de 10 min de exposición, la temperatura disminuirá de forma automática bajo el control de la rutina de LabVIEW. </li><li> Observar la forma de los cristales a medida que disminuye la temperatura. En algún momento, la ráfaga repentina de los cristales de hielo pueden ser observados. La temperatura a la que ocurre esto se observa como la temperatura ráfaga cristal. </li><li> El uso de análisis de vídeo para determinar el punto de fusión y la temperatura precisa de ráfaga. En primer lugar, mediante el uso de análisis de vídeo, encontrar el punto de fusión exacto. Recordemos que la temperatura más alta a la que ha dejado de fusión se determina que el punto de fusión. Documentar este punto de fusión en un programa de hoja de cálculo. Entonces, determinar la temperatura exacta cristal ráfaga, y documentar este valortambién. La diferencia entre el punto de fusión y el punto de congelación, o la temperatura ráfaga de cristal, es la actividad de histéresis térmica de la solución de IBP. </li></ol> 4. Medida de la actividad TH en función del tiempo <ol><li> Seguir el protocolo descrito en las Secciones 3.1-3.3 para preparar un solo cristal de hielo. </li><li> Después de la formación de los cristales, ajuste el tiempo de retardo de la rampa como desee y, a su vez en la rampa. </li><li> La temperatura disminuye a una tasa fija (de acuerdo con los requisitos de los operadores) de forma automática una vez que el tiempo de retardo de rampa ha pasado. </li><li> Documentar la temperatura a la que se produce la ráfaga de cristal. Calcular el tiempo de exposición (el tiempo entre la formación de cristales y la ráfaga de cristal). </li><li> Repetir el experimento para tiempos de retardo diferentes y representar gráficamente la actividad TH como una función del tiempo de exposición para evaluar la dependencia del tiempo de la actividad TH. </li></ol>

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No hay conflictos de interés declarado.

Este trabajo demuestra el funcionamiento de un osmómetro de nanolitros controlado por computadora que permite mediciones precisas de la actividad TH con control de temperatura extraordinaria. En cualquier sistema sensible a la temperatura, gradientes de temperatura no deseados deben ser evitados. Para evitar gradientes de temperatura en el aparato que aquí se presenta, la gota de solución de ensayo debe ser posicionado en el centro de un agujero en la etapa de refrigeración de cobre del disco (paso 2,7). Adicionalmente, el monocristal debe estar en el centro de la gotita en lugar de cerca de los bordes (en la mayoría de los casos, esto sucederá de forma espontánea). La dependencia del tiempo descrito indica que la velocidad de enfriamiento puede influir en las lecturas de TH. Por lo tanto, se sugiere incluir un informe del tiempo durante el cual el cristal fue expuesta a la solución antes de la refrigeración, así como la velocidad de enfriamiento. Normalmente nos esperó 10 min antes de la rampa descendente de la temperatura a 0,01 ° C pasos cada sec 4. Los co-LabVIEW controladosetapa Oling fue adaptado para uso con un microscopio invertido en el que los dispositivos de microfluidos podría ser térmicamente manipulado. Este sistema facilita la realización de experimentos de intercambio de soluciones que implican cristales de hielo y IBPS etiquetados con eGFP 9, 10, 16. El sistema LabVIEW controlado puede ser adaptado a una etapa Clifton conectando el controlador de temperatura 3040 a través de un circuito de adaptación designado eléctrica. Tal sistema es operado en el laboratorio Davies 17. El software LabVIEW y el diseño de la adaptación designado circuito eléctrico para la etapa de Clifton están disponibles bajo petición. En conclusión, se describe un osmómetro de nanolitros que facilita el control sensible y manipulación de la temperatura y la tasa de aumento de la temperatura y disminución (con 0,002 ° C sensibilidad), coordinado con un interfaz de vídeo a través de una rutina de LabVIEW para análisis en tiempo real. Este sistema puede realizar reproducibles de velocidad controlada experimentos que son Important para investigar la cinética de la interacción IBP con hielo. Tales experimentos pueden abordar varios largamente debatido cuestiones relacionadas con el mecanismo de acción de los IBP.

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> Número de catálogo / modelo </td><td> Comentarios </td></tr><tr><td> Aceite de inmersión tipo B </td><td> Cargille Laboratorios </td><td> 16484 </td><td></td></tr><tr><td> Drierite </td><td> WA Hammond Drierite </td><td> 043063 2270g </td><td></td></tr><tr><td> Micro 90 solución de limpieza </td><td> Cole-Parmer </td><td> <a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18100-11</a> </td><td></td></tr><tr><td> Capilar extractor </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> Tubos capilares de cristal </td><td> Marca GnbH Persona </td><td> 7493 21 </td><td> 75 mm de largo, 1,15 de diámetro </td></tr><tr><td> Temperaturacontrolador </td><td> Newport, Irvine, California, Estados Unidos </td><td> Modelo 3040 </td><td> Modelo 3040 </td></tr><tr><td> Microscopio de luz </td><td> Olimpo </td><td> Modelo BH2 </td><td></td></tr><tr><td> Objetivo de 10x </td><td> Olimpo </td><td></td><td> S Plan 10, 0,3, 160/0.17 </td></tr><tr><td> Objetivo 50x </td><td> Nikon </td><td></td><td> CF plan, 50X/0.55 EPI ELWD </td></tr><tr><td> Cámara CCD </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tubos Tygon </td><td> Saint-Gobain, París, Francia </td><td></td><td> Formulación Tygon S-50-HL Tubería </td></tr><tr><td> Vidrio de la jeringa (2 ml) </td><td> Poulten-Graf, Wertheim, Alemania </td><td> 7 10227 </td><td></td></tr><tr><td> Tarjeta PCI GPIB- </td><td> National Instruments, Austin, Texas, EE.UU. </td><td> 778032-01 </td><td></td></tr><tr><td> Vídeo marco grabBER IMAQ PCI-1407 </td><td> National Instruments, Austin, Texas, EE.UU. </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW System Software Design </td><td> National Instruments, Austin, Texas, EE.UU. </td><td> Versión 8 </td><td></td></tr><tr><td> Software DivX Author </td><td> DiVx LLC, San Diego, CA, EE.UU. </td><td></td><td></td></tr></tbody></table>

labview-operated novel nanoliter osmometer for ice binding protein investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

La medición de la dependencia del tiempo TH El osmómetro LabVIEW operado nanolitros facilita la realización de mediciones precisas de la actividad TH. La tasa de reducción de temperatura constante permite la medición de la dependencia del tiempo TH. El control preciso de la temperatura activado por el osmómetro de nanolitros era crucial para estos experimentos. El tiempo de exposición de un cristal de hielo a los IBP en solución se define como el periodo de tiempo desde la formación del cristal (el final del proceso de fusión) hasta que el crecimiento repentino de hielo alrededor del cristal (cristal ráfaga). Se encontró que el tiempo de exposición de los cristales de hielo a los IBPs afectado de forma crucial la actividad TH. Los períodos cortos de exposición a IBP (unos pocos segundos) produjo una actividad TH baja en el Mp IBP-GFP solución (2,4 mM) (Figura 5). La actividad TH aumentó con el tiempo de exposición IBP hasta que alcanzó una meseta a 4 min de exposición IBP. A concentraciones más altas de IBP, la placaau se alcanzó a los tiempos más cortos. <img alt="Figura 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> Figura 1. IBPs diagrama esquemático que ilustra adsorbido en hielo. Aprobada con permiso de 10. <img alt="Figura 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> Figura 2. La etapa de enfriamiento. A) conectado a los tubos en el microscopio. B) Sin el liderazgo superior. C) Diagrama esquemático. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="Figura 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> Figura 3. Captura de pantalla de la interfaz de LabVIEW. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Cl</a>ick aquí para ampliar la figura. <img alt="Figura 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> Figura 4. Gráfico de temperatura estabilidad. El controlador de temperatura se ajustó a bajar la temperatura de 0,01 ° C cada 15 seg. <img alt="Figura 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> Figura 5. Mp actividad TH IBP como una función del tiempo de exposición de cristales de hielo a los IBP. Cada punto de tiempo es la media de 3-6 experimentos.

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Proteínas de unión de hielo (IBP), también conocidas como proteínas anticongelantes, inhibir el crecimiento del hielo y son un aditivo prometedor para su uso en la crioconservación de tejidos. La principal herramienta utilizada para investigar los IBP es el osmómetro nanolitros. Hemos desarrollado un hogar diseñado etapa de enfriamiento montado en un microscopio óptico y se controla utilizando una rutina de costumbre-construido LabVIEW. El osmómetro nanoliter descrito aquí manipulado la temperatura de la muestra de una manera ultra-sensible.

LabVIEW operado Novela Osmometer nanolitros de Investigaciones de hielo de fijación con proteínas

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

Research

JoVE Journal

Chemistry

20.3K vistas.  The Hebrew University of Jerusalem. Proteínas de unión de hielo (IBP), también conocidas como proteínas anticongelantes, inhibir el crecimiento del hielo y son un aditivo prometedor para su uso en la crioconservación de tejidos. La principal herramienta utilizada para investigar los IBP es el osmómetro nanolitros. Hemos desarrollado un hogar diseñado etapa de enfriamiento montado en un microscopio óptico y se controla utilizando una rutina de costumbre-construido LabVIEW. El osmómetro nanoliter des...

Video: LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

La pasivación de la superficie de Estudios sola proteína-molécula

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

Investigación

Química

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Unión covalente de BMP-2 en las superficies utilizando una monocapa enfoque de auto-ensamblada

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

La Determinación De Los Planos De Unión Al Hielo De Las Proteínas Anticongelante Por La Afinidad De Plano De Hielo Basado En Fluorescencia

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

Interferometría Bio-capa para Cinética de interacciones proteína-proteína y alostéricos Efectos ligando medición

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Iridium (III) luminiscentes Sonda para la detección de la proteína malaria biomarcador histidina Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Emulsiones agua en aceite: Un nuevo sistema para el ensamblaje de las proteínas de unión de clorofila-solubles en agua con pigmentos hidrófobos

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

Un ELISA basado Encuadernación y método Competencia determinación rápida interacciones ligando-receptor

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE - eluida-gel transparente nativo fracción líquida atrapamiento de electroforesis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Las investigaciones en el complejo Ga (III) de EOB-DTPA y su<sup&gt; 68</sup&gt; Ga radiomarcados analógica

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

Nuevas técnicas para la observación de la dinámica estructural de los cristales líquidos Photoresponsive

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...