Cette recherche a été financée par l&#39;ISF, la NSF, et l&#39;ERC. Nous tenons à souligner l&#39;aide technique avec le niveau de température de Randy Milford, Michael Koren, Doug Shafer, et Jeremy Dennison. Assistance au développement de logiciels ont été fournis par Ou Chen, Xu Di, Rajesh Sannareddy, et Sumit Bhattachary. Nous tenons à remercier nos collaborateurs Prof Peter L. Davies et Dr Laurie A. Graham pour la protéine de fusion IBP et des discussions utiles. Nous remercions également les membres de laboratoire Dr. Maya Bar-Dolev, Yangzhong Qin, le Dr Yeliz Celik, le Dr Natalya Pertaya, Ortal Mizrahy, et Guy Shlomit pour leur rétroaction des utilisateurs.

0. Procédures préliminaires <ol><li> Capillaire en verre pour injection de solution. Aide d&#39;un extracteur capillaire (Narishige, Tokyo, Japon), préparer une pipette marquée avec une ouverture bien à partir d&#39;un tube de verre capillaire micro (Marque GMBH, Wertheim, Allemagne). La taille de l&#39;ouverture doit être vérifiée par le passage d&#39;air à travers le capillaire d&#39;obtenir bulles fines à l&#39;eau claire. Si le capillaire est fermé, alors on peut l&#39;ouvrir en brisant son bord. Cela peut être accompli en appuyant sur ou de la rayer doucement contre l&#39;eau contenant parois du tube. Préparer le capillaire telle que l&#39;ouverture est pratiquement bloquée, mais est suffisamment ouvert pour permettre la formation de bulles sous-millimétriques. </li><li> Nettoyage disque de cuivre. Soniquer les disques de cuivre pendant 10 min dans 0,1% Micro-90 savon (Cole-Parmer, Vernon Hills, Illinois, USA), puis laver avec de l&#39;eau bidistillée. Introduire les disques dans un mélange isopropanol solution (technique) et soniquer à nouveau pendant 10 min. Nageoireallié, sécher les disques à l&#39;aide de l&#39;air filtré. Cette étape de nettoyage est essentiel pour éviter la contamination IBP entre les expériences. </li><li> Assemblage lamelle double couche. Un assemblage lamelle a été préparé pour permettre une observation de l&#39;échantillon, sans condensation de l&#39;humidité sur la surface du verre de couverture. Ceci a été réalisé en plaçant un Drierite (WA Hammond Drierite, Xenia, Ohio, Etats-Unis) de particules (2 mm de diamètre) entre deux lamelles qui ont ensuite été collés avec un pistolet à colle chaude. Cette configuration empêche la condensation qui pourrait bloquer la vue lorsque l&#39;échantillon a été refroidi à des températures basses et supprimé la nécessité de souffler de l&#39;air sec sur la fenêtre d&#39;observation. </li></ol> 1. Phase de refroidissement Set-up <ol><li> Connectez l&#39;entrée du flux de l&#39;eau et de sortie de l&#39;étape de refroidissement à 4 mm de diamètre intérieur des tubes Tygon (Saint-Gobain, Paris, France), et connecter le tube d&#39;entrée du débit d&#39;eau à une pompe à eau. </li><li> Connecter un tube intérieur 4 mm de diamètre Tygon à l&#39;entrée de l&#39;étage de refroidissement to fournir de l&#39;air sec. L&#39;air est séché en utilisant une colonne en ligne Drierite. </li><li> Actionner les pompes à air et de l&#39;eau. Notez que les éléments de refroidissement ne doit pas être exécuté sans un dissipateur de chaleur. </li><li> Allumez le régulateur de température, appareil photo, et la routine LabVIEW. </li></ol> 2. Préparation des échantillons <ol><li> Placez une goutte de 4.3 pi d&#39;huile d&#39;immersion B (Cargille laboratoires, Cedar Grove, New Jersey, Etats-Unis) sur la face arrière d&#39;un disque de cuivre de diamètre 7 mm avec 500 trous forés pm à travers le disque. </li><li> Placez le disque de cuivre sur la scène de refroidissement avec le côté huile d&#39;immersion vers le bas. </li><li> Raccorder le tube capillaire (le bord émoussé) pour un diamètre de 0,7 mm Tygon tube intérieur relié à l&#39;autre extrémité sur une seringue 2 ml en verre (Poulten-Graf, Wertheim, Allemagne). </li><li> Avant d&#39;utiliser le tube capillaire, vérifier la petite ouverture du capillaire de sorte que l&#39;ouverture est d&#39;une taille appropriée (voir les procédures préliminaires). </li><li> Insérez lentement la glaart capillaire dans le tube d&#39;échantillon préparé IBP protéines (2,4 uM Mp IBP-GFP dans 20 mM de CaCl2 et 25 mM de Tris-HCl à pH 8, voir la référence 10 pour les détails de préparation) et retirer la seringue en verre jusqu&#39;à ce que le capillaire en verre contient 0,1 pl de la solution de protéine. </li><li> Commencez l&#39;enregistrement vidéo via le logiciel LabVIEW. </li><li> Insérer la pointe dans le capillaire en verre (contenant la solution de protéine) dans l&#39;un des trous dans le disque de cuivre sur l&#39;étage de refroidissement. </li><li> Tout en observant au microscope (Olympus, Tokyo, Japon, objectif 10x), soigneusement pénétrer dans la couche d&#39;huile d&#39;immersion avec la pointe capillaire en verre, puis appuyez sur la seringue en verre (très délicatement) afin de fournir une petite quantité (environ 10 nl) de la protéine solution pour créer une gouttelette à 200 um. </li><li> Couvrez le trou dans la phase de refroidissement avec l&#39;ensemble lamelle double couche (voir les procédures préliminaires). </li></ol> 3. Mesure de l&#39;activité TH <ol><li> Préss sur le bouton de refroidissement et régler la température à -40 ° C. </li><li> Initialement, la gouttelette solution doit être limpide. A basse température, typiquement dans la gamme -30 ° C à -35 ° C, la couleur des gouttelettes changements, ce qui indique que la solution a été congelé. Immédiatement après le prélèvement a gelé, augmenter la température lentement jusqu&#39;à ce que la glace commence à fondre en vrac. Une augmentation progressive de la température est nécessaire pour éviter le dépassement de la température qui pourrait se traduire par la fusion complète de l&#39;échantillon. </li><li> Basculer vers un objectif 50x et commencer à faire fondre la glace en réglant la température. Ce réglage est interactif, et les étapes finales sont généralement effectuées à l&#39;aide des mesures de température petits de 0,002 ° C. Continuer à faire fondre jusqu&#39;à ce qu&#39;un monocristal reste. La taille finale du cristal doit être d&#39;environ 10 um. La plus haute température à laquelle la fusion a cessé est déterminé comme étant le point de fusion est déterminé avec précision et dans l&#39;étape d&#39;analyse vidéo plus tard. </li> &lt;li&gt; Régler la température à quelques centièmes de degré Celsius en dessous du point de fusion du cristal et de commencer une rampe de température avec un retard de 10 min. Ajuster le taux de rampe comme vous le souhaitez. Pendant ce temps, le cristal sera exposée aux IBP. </li><li> À la fin du temps de 10 min d&#39;exposition, la température diminuera automatiquement sous le contrôle de la routine LabVIEW. </li><li> Observer la forme cristalline que la température diminue. À un certain moment, l&#39;explosion soudaine du cristal de glace peuvent être observés. La température à laquelle cela se produit est à noter que la température éclat de cristal. </li><li> Utiliser l&#39;analyse vidéo afin de déterminer le point de fusion et la température exacte d&#39;éclatement. Tout d&#39;abord, en utilisant l&#39;analyse vidéo, trouver le point de fusion précis. Rappelons que la plus haute température à laquelle la fusion a cessé est déterminé comme étant le point de fusion. Documentez ce point de fusion dans un tableur. Ensuite, déterminer la température du cristal précise rafale, et documenter cette valeurainsi. La différence entre le point de fusion et le point de congélation, ou la température éclatement de cristal, est l&#39;activité d&#39;hystérésis thermique de la solution IBP. </li></ol> 4. Mesure de l&#39;activité TH en fonction du temps <ol><li> Suivre le protocole décrit dans les sections 3.1 à 3.3 pour préparer un monocristal de glace. </li><li> Après la formation de la glace, le temps de retard de la rampe comme vous le souhaitez, et tourner sur la rampe. </li><li> La température sera réduite à un taux fixe (en fonction des besoins des opérateurs) automatiquement une fois le temps de retard rampe est passée. </li><li> Documenter la température à laquelle se produit la rupture de cristal. Calculer le temps d&#39;exposition (le temps entre la formation des cristaux et la salve de cristal). </li><li> Répéter l&#39;expérience pour divers temps de retard et tracer l&#39;activité TH en fonction de la durée d&#39;exposition pour évaluer la dépendance temporelle de l&#39;activité TH. </li></ol>

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L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 1309-1314 (2013).</li><li>Middleton, A. J., Marshall, C. B., Faucher, F., Bar-Dolev, M., Braslavsky, I., Campbell, R. L., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+protein+from+freeze-tolerant+grass+has+a+beta-roll+fold+with+an+irregularly+structured+ice-binding+site.">Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.</a> J. Mol. Biol. 416, 713-724 (2012).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Ce travail démontre le fonctionnement d&#39;un osmomètre nanolitre commandé par ordinateur qui permet des mesures précises de l&#39;activité TH avec contrôle de la température extraordinaire. Dans tout système sensible à la température, les gradients de température indésirables doivent être évitées. Afin d&#39;éviter des gradients de température dans l&#39;appareil présenté ici, la gouttelette de solution d&#39;essai doit être positionné au centre d&#39;un trou dans la phase de refroidissement en cuivre disque (étape 2,7). En outre, le cristal unique devrait être au centre de la goutte plutôt que près des bords (dans la plupart des cas, cela se produira spontanément). La dépendance temporelle décrite indique que la vitesse de refroidissement peuvent influencer les lectures TH. Ainsi, on propose notamment un rapport de la durée pendant laquelle le cristal a été exposée à la solution avant le refroidissement, ainsi que la vitesse de refroidissement. En général, nous a attendu 10 minutes avant de la décélération de la température de 0,01 ° C chaque étapes 4 sec. Les co-contrôlées LabVIEWOling étape a été adapté pour être utilisé avec un microscope inversé sur lequel des dispositifs microfluidiques peuvent être manipulés thermiquement. Ce système facilite la réalisation d&#39;expériences d&#39;échange de solutions impliquant des cristaux de glace et IBPS taggés avec eGFP 9, 10, 16. Le système à commande LabVIEW peut être adapté à un stade Clifton en connectant le contrôleur de température 3040 par l&#39;intermédiaire d&#39;un circuit électrique désigné adaptation. Un tel système est utilisé dans le laboratoire Davies 17. Le logiciel LabVIEW et l&#39;adaptation désigné conception de circuit électrique pour la phase de Clifton sont disponibles sur demande. En conclusion, nous décrivons un osmomètre nanolitre qui facilite le contrôle et la manipulation sensible de la température et le taux d&#39;augmentation de la température et une diminution (avec 0,002 ° C de sensibilité), en coordination avec une interface vidéo à travers une routine LabVIEW pour analyse en temps réel. Ce système peut effectuer reproductibles taux contrôlées par des expériences qui sont important pour enquêter sur la cinétique des interactions IBP avec de la glace. De telles expériences peuvent répondre à plusieurs questions débattues à long entourant le mécanisme d&#39;action de IBP.

<table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue / modèle </td><td> Commentaires </td></tr><tr><td> Huile à immersion de type B </td><td> Laboratoires Cargille </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 solution de nettoyage 90 </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> Capillaire extracteur </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> Tubes capillaires en verre </td><td> Marque GNBH </td><td> 7493 21 </td><td> 75 mm de long, 1,15 de diamètre </td></tr><tr><td> Températurecontrôleur </td><td> Newport, Irvine, Californie, États-Unis </td><td> Modèle 3040 </td><td> Modèle 3040 </td></tr><tr><td> Microscope optique </td><td> Olympe </td><td> Modèle BH2 </td><td></td></tr><tr><td> Objectif 10x </td><td> Olympe </td><td></td><td> S Plan de 10, 0,3, 160/0.17 </td></tr><tr><td> Objectif 50x </td><td> Nikon </td><td></td><td> CF plan, 50X/0.55 PEV ELWD </td></tr><tr><td> Caméra CCD </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tubes Tygon </td><td> Saint-Gobain, Paris, France </td><td></td><td> Formulation Tygon S-50-HL tubes </td></tr><tr><td> Seringue en verre (2 ml) </td><td> Poulten-Graf, Wertheim, Allemagne </td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-PCI </td><td> National Instruments, Austin, Texas, USA </td><td> 778032-01 </td><td></td></tr><tr><td> Vidéo cadre grabBER IMAQ PCI-1407- </td><td> National Instruments, Austin, Texas, USA </td><td> 322156B-01 </td><td></td></tr><tr><td> Logiciel LabVIEW System Design </td><td> National Instruments, Austin, Texas, USA </td><td> Version 8 </td><td></td></tr><tr><td> Logiciel DivX Author </td><td> DiVx LLC, San Diego, CA, USA </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).

Mesure de la dépendance temporelle TH L&#39;osmomètre nanolitre LabVIEW fonctionnant facilite l&#39;exécution des mesures précises de l&#39;activité TH. Le taux de réduction à température constante permet la mesure de la dépendance temporelle TH. Le contrôle précis de la température activée par l&#39;osmomètre nanolitre était crucial pour ces expériences. Le temps d&#39;exposition d&#39;un cristal de glace à l&#39;IBP en solution est définie comme étant la période de temps à partir de la formation de la glace (la fin du processus de fusion) jusqu&#39;à ce que la croissance brusque de la glace autour du cristal (cristaux rafale). Nous avons constaté que le temps d&#39;exposition des cristaux de glace à l&#39;IBP cruciale affecté l&#39;activité TH. De courtes périodes d&#39;exposition IBP (quelques secondes) produit une activité TH bas dans le Mp IBP-GFP solution (2,4 uM) (Figure 5). L&#39;activité TH augmente avec le temps d&#39;exposition IBP jusqu&#39;à ce qu&#39;il atteigne un plateau à 4 min IBP exposition. À des concentrations plus élevées IBP, la plaqueau a été atteint à des temps plus courts. <img alt="Figure 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> IBP Figure 1. Schéma illustrant adsorbé sur de la glace. Adoptée avec la permission de 10. <img alt="Figure 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> Figure 2. L&#39;étape de refroidissement. A) reliés à des tubes sur le microscope. B) Sans plomb supérieure. Schéma C). <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="Figure 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> Figure 3. Capture d&#39;écran de l&#39;interface de LabVIEW. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Cl</a>ick ici pour agrandir la figure. <img alt="Figure 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> Figure 4. Graphique Stabilité de la température. Le régulateur de température a été fixé pour abaisser la température de 0,01 ° C toutes les 15 secondes. <img alt="Figure 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> Figure 5. Activité Mp TH IBP en fonction du temps d&#39;exposition pour les cristaux de glace IBP. Chaque point est la moyenne de temps de 3-6 expériences.

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

Protéines de liaison de glace (IBP), également appelées protéines antigel, inhiber la croissance de la glace et sont un additif prometteur pour une utilisation dans la cryoconservation des tissus. Le principal outil utilisé pour étudier IBP est le osmomètre nanolitre. Nous avons développé une maison conçue étape de refroidissement monté sur un microscope optique et contrôlé à l&#39;aide d&#39;une routine sur mesure LabVIEW. L&#39;osmomètre nanolitre décrit ici manipulé la température de l&#39;échantillon d&#39;une manière ultra-sensible.

LabVIEW fonctionnant Osmomètre nanolitre Novel des enquêtes de glace Binding Protein

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 vues.  The Hebrew University of Jerusalem. Protéines de liaison de glace (IBP), également appelées protéines antigel, inhiber la croissance de la glace et sont un additif prometteur pour une utilisation dans la cryoconservation des tissus. Le principal outil utilisé pour étudier IBP est le osmomètre nanolitre. Nous avons développé une maison conçue étape de refroidissement monté sur un microscope optique et contrôlé à l'aide d'une routine sur mesure LabVIEW. L'osmomètre nanolitre décri...

Vidéo: 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.

passivation de surface pour les études de protéines unique molécule

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

Recherche

Chimie

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.

Liaison covalente de BMP-2 sur les surfaces en utilisant une approche monocouche auto-assemblée

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.

Détermination des plans de liaison à la glace des protéines antigel par affinité du plan de glace basée sur la fluorescence

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.

Bio-couche interférométrie pour Cinétique des interactions protéine-protéine et allostériques Effets ligand mesure

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) luminescent sonde pour la détection de la protéine riche en histidine Malarial biomarqueurs protéines-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.

L&#39;eau dans l&#39;huile Emulsions: un nouveau système d&#39;assemblage des protéines Chlorophylle liaison solubles dans l&#39;eau avec Pigments hydrophobes

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 à base de liaison et la méthode de la concurrence de déterminer rapidement interactions ligand-récepteur

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 - Clear natif Gel-élué Fraction liquide Entrapment Electrophoresis

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.

Enquêtes sur le complexe Ga (III) de EOB-DTPA et son<sup&gt; 68</sup&gt; Ga radiomarqué analogique

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.

Nouvelles Techniques d’observation dynamique des structures des cristaux liquides photopériode

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