Questa ricerca è stata sostenuta dalla ISF, NSF, e ERC. Vorremmo riconoscere aiuto tecnico con la fase di temperatura da Randy Milford, Michael Koren, Doug Shafer, e Jeremy Dennison. Assistenza allo sviluppo del software è stato fornito da o Chen, Di Xu, Rajesh Sannareddy e Sumit Bhattachary. Vorremmo ringraziare i nostri collaboratori il Prof. Peter L. Davies e il dottor Laurie A. Graham per la proteina Mp IBP e utili discussioni. Ringraziamo anche i membri del laboratorio Dr. Maya Bar-Dolev, Yangzhong Qin, il dottor Yeliz Celik, il dottor Natalya Pertaya, Ortal Mizrahy, e Guy Shlomit per il loro feedback degli utenti.

0. Procedure preliminari <ol><li> Capillare di vetro per l&#39;iniezione soluzione. Utilizzando un estrattore capillare (Narishige, Tokyo, Giappone), preparare una pipetta con una forte apertura multa da un tubo capillare di vetro micro (Marca GMBH, Wertheim, Germania). La dimensione dell&#39;apertura deve essere verificata facendo passare aria attraverso il capillare per ottenere bubbling bene in acqua pulita. Se il capillare è chiuso, si può aprirlo rompendo il bordo. Ciò può essere realizzato per stampaggio o graffiare delicatamente contro l&#39;acqua contenente pareti del tubo. Preparare il capillare tale che l&#39;apertura è quasi bloccata, ma sufficientemente aperta per consentire la formazione di bolle sub-millimetri. </li><li> Pulizia del disco di rame. Sonicare i dischi di rame per 10 min a 0,1% Micro-90 sapone (Cole-Parmer, Vernon Hills, Illinois, Stati Uniti d&#39;America), poi lavare con acqua bidistillata. Introdurre i dischi in un (tecnico) soluzione di isopropanolo e sonicare ancora per 10 min. Pinnaalleato, asciugare i dischi con aria filtrata. Questa fase di pulizia è fondamentale per evitare la contaminazione tra esperimenti IBP. </li><li> Doppio strato di montaggio coprioggetti. Gruppo coprioggetti è stato preparato per consentire l&#39;osservazione del campione senza condensa di umidità sulla superficie del vetro di copertura. Questo è stato ottenuto inserendo una (WA Hammond Drierite, Xenia, Ohio, USA) Drierite particelle (2 mm di diametro) tra due vetrini che poi sono stati incollati con una pistola per colla a caldo. Questa configurazione impedisce condensa che potrebbe bloccare la vista quando il campione è stato raffreddato a basse temperature e rimosso la necessità di soffiare aria secca sulla finestra di osservazione. </li></ol> 1. Fase di raffreddamento Set-up <ol><li> Collegare l&#39;ingresso del flusso d&#39;acqua e di uscita della fase di raffreddamento a 4 mm di diametro interno tubi Tygon (Saint-Gobain, Parigi, Francia), e collegare il tubo di flusso dell&#39;acqua in ingresso ad una pompa dell&#39;acqua. </li><li> Collegare un tubo interno 4 mm di diametro Tygon all&#39;ingresso del t stadio di raffreddamentoo fornire aria secca. L&#39;aria è stata essiccata con un in-linea di colonna Drierite. </li><li> Far funzionare le pompe di aria e acqua. Si noti che gli elementi di raffreddamento non dovrebbe essere eseguito senza un dissipatore di calore. </li><li> Accendere il regolatore di temperatura, macchina fotografica, e di routine LabVIEW. </li></ol> 2. Preparazione del campione <ol><li> Posizionare una gocciolina 3-4 pl di olio B immersione (Cargille laboratori, Cedar Grove, New Jersey, USA) sul lato posteriore di un disco diametro 7 mm rame avente fori 500 micron perforati attraverso il disco. </li><li> Posizionare il disco di rame sulla fase di raffreddamento con il lato rivolto verso il basso immersione in olio. </li><li> Collegare il tubo capillare (il bordo smussato) ad un tubo di diametro interno 0,7 millimetri Tygon collegata all&#39;altra estremità ad una siringa 2 ml di vetro (Poulten-Graf, Wertheim, Germania). </li><li> Prima di utilizzare il tubo capillare, controllare la piccola apertura del capillare per garantire che l&#39;apertura è una dimensione appropriata (vedere le procedure preliminari). </li><li> Inserire lentamente il GLAss capillare nella provetta preparato proteina IBP (2,4 pM Mp IBP-GFP in 20 mM CaCl 2 e 25 mM Tris-HCl a pH 8, vedi riferimento 10 per i dettagli di preparazione) e tirare la siringa di vetro fino a quando il capillare di vetro contiene 0,1 pl della soluzione proteica. </li><li> Iniziare la registrazione video tramite il software LabVIEW. </li><li> Inserire il bordo tagliente del capillare di vetro (contenente la soluzione di proteina) in uno dei fori del disco di rame in fase di raffreddamento. </li><li> Osservando al microscopio (Olympus, Tokyo, Giappone, obiettivo 10x), con attenzione penetrare lo strato di olio di immersione con la punta capillare di vetro, e premere la siringa di vetro (molto delicatamente) per fornire una piccola quantità (~ 10 nl) della proteina soluzione per creare un droplet 200 micron. </li><li> Coprire il foro nella fase di raffreddamento con il gruppo a doppio strato coprioggetto (consultare le procedure preliminari). </li></ol> 3. TH attività di misura <ol><li> Press il pulsante di raffreddamento e la temperatura a -40 ° C. </li><li> Inizialmente, la goccia soluzione sarà limpida. A basse temperature, tipicamente nel campo da -30 ° C a -35 ° C, il colore gocciolina cambia, indicando che la soluzione è stata congelata. Immediatamente dopo che il campione è congelato, aumentare la temperatura lentamente finché il ghiaccio bulk inizia a fondere. Un graduale aumento della temperatura è necessaria per evitare il superamento della temperatura che potrebbe portare alla completa fusione del campione. </li><li> Passare a un obiettivo 50x e cominciare a sciogliere il ghiaccio con regolazione della temperatura. Questa regolazione è interattivo, e le fasi finali sono in genere eseguita con passi piccoli temperatura di 0,002 ° C. Continuare a fondere fino a quando rimane un singolo cristallo. La dimensione finale del cristallo deve essere di circa 10 micron. La massima temperatura alla quale ha cessato di fusione è determinato essere il punto di fusione ed è determinata con precisione nella fase successiva analisi video. </li> &lt;li&gt; Impostare la temperatura di pochi centesimi di un grado Celsius sotto del punto di fusione del cristallo e iniziare una rampa di temperatura con un ritardo di 10 min. Regolare la velocità di rampa come desiderato. Durante questo tempo, il cristallo viene esposto ai IBPs. </li><li> Al termine del tempo di 10 minuti di esposizione, la temperatura diminuisce automaticamente sotto il controllo della routine LabVIEW. </li><li> Osservare la forma di cristallo al diminuire della temperatura. Ad un certo punto, l&#39;improvviso scoppio del cristallo di ghiaccio può essere osservato. La temperatura alla quale si verifica ciò è notato come la temperatura scoppio cristallo. </li><li> Utilizzare video analisi per determinare il punto preciso fusione e la temperatura di scoppio. In primo luogo, con l&#39;analisi video, trovare il punto esatto di fusione. Ricordiamo che la temperatura massima alla quale ha cessato di fusione è determinato essere il punto di fusione. Documentare questo punto di fusione in un programma di foglio di calcolo. Quindi, determinare la precisa temperatura scoppio cristallo, e documentare questo valorepure. La differenza tra il punto di fusione e il punto di congelamento, o temperatura scoppio cristallo, è l&#39;attività isteresi termica della soluzione IBP. </li></ol> 4. Misura della funzione del tempo di attività TH <ol><li> Seguire il protocollo descritto nelle sezioni 3,1-3,3 per preparare un singolo cristallo di ghiaccio. </li><li> Dopo la formazione del cristallo, impostare il tempo di ritardo della rampa nel modo desiderato, e accendere la rampa. </li><li> La temperatura diminuirà ad un tasso fisso (a seconda delle esigenze degli operatori) automaticamente una volta che il tempo di ritardo rampa è passato. </li><li> Documentare la temperatura alla quale si verifica la rottura di cristallo. Calcolare il tempo di esposizione (tempo tra la formazione di cristalli e lo scoppio cristallo). </li><li> Ripetere l&#39;esperimento per diversi tempi di ritardo e tracciare l&#39;attività TH come funzione del tempo di esposizione per valutare la dipendenza dal tempo dell&#39;attività TH. </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+adaptation+in+Arctic+and+Antarctic+fungi.">Cold adaptation in Arctic and Antarctic fungi.</a> New Phytol. 151, 341-353 (2001).</li><li>Gilbert, J. A., Hill, P. J., Dodd, C. E., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+antifreeze+protein+activity+in+Antarctic+lake+bacteria.">Demonstration of antifreeze protein activity in Antarctic lake bacteria.</a> Microbiology. 150, 171-180 (2004).</li><li>Raymond, J. A., DeVries, A. 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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=Superheating+of+ice+crystals+in+antifreeze+protein+solutions.">Superheating of ice crystals in antifreeze protein solutions.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5423-5428 (2010).</li><li>Gilbard, J. P., Farris, R. L., Santamaria, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolarity+of+tear+microvolumes+in+keratoconjunctivitis+sicca.">Osmolarity of tear microvolumes in keratoconjunctivitis sicca.</a> Arch. Ophthalmol. 96, 677-681 (1978).</li><li>Gilbert, J. A., Davies, P. L., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperactive,+Ca2+-dependent+antifreeze+protein+in+an+Antarctic+bacterium.">A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium.</a> FEMS Microbiol. Lett. 245, 67-72 (2005).</li><li>Soriano, J., Braslavsky, I., Xu, D., Krichevsky, O., Stavans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universality+of+persistence+exponents+in+two-dimensional+ostwald+ripening.">Universality of persistence exponents in two-dimensional ostwald ripening.</a> Phys. Rev. Lett. 103, (2009).</li><li>Tomczak, M. M., Marshall, C. B., Gilbert, J. A., 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=A+facile+method+for+determining+ice+recrystallization+inhibition+by+antifreeze+proteins.">A facile method for determining ice recrystallization inhibition by antifreeze proteins.</a> Biochem. Bioph. Res. Co. 311, 1041-1046 (2003).</li><li>Knight, C. A., Hallett, J., Devries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+Effects+on+Ice+Recrystallization+-+an+Assessment+Technique.">Solute Effects on Ice Recrystallization - an Assessment Technique.</a> Cryobiology. 25, 55-60 (1988).</li><li>Celik, Y., Drori, R., Pertaya-Braun, N., Altan, A., Barton, T., Bar-Dolev, M., Groisman, A., Davies, P. 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>

Nessun conflitto di interessi dichiarati.

Questo lavoro illustra il funzionamento di un computer controllato osmometro nanolitri che permette misure accurate di attività TH con controllo della temperatura straordinario. In qualsiasi sistema sensibile alla temperatura, gradienti di temperatura desiderati devono essere evitati. Per evitare gradienti di temperatura nell&#39;apparato qui presentato, la gocciolina di soluzione campione deve essere posizionato al centro di un foro nella fase di raffreddamento in rame disco (punto 2.7). Inoltre, il singolo cristallo dovrebbe essere al centro della goccia piuttosto che vicino ai bordi (nella maggior parte dei casi, ciò avverrà spontaneamente). La dipendenza dal tempo descritto indica che la velocità di raffreddamento può influenzare le letture TH. Pertanto, si suggerisce una relazione tra il tempo durante il quale il cristallo è stato esposto alla soluzione prima di raffreddamento, così come la velocità di raffreddamento. Abbiamo tipicamente aspettato 10 min prima rampa di discesa della temperatura a 0,01 ° C passaggi ogni 4 sec. I LabVIEW controllati cofase oling stato adattato per l&#39;uso con un microscopio invertito su cui dispositivi microfluidici potrebbe essere termicamente manipolato. Tale sistema facilita l&#39;esecuzione di esperimenti di cambio soluzione con cristalli di ghiaccio e IBPs con etichetta eGFP 9, 10, 16. Il LabVIEW sistema controllato può essere adattata ad una fase Clifton collegando il regolatore di temperatura 3040 tramite un circuito di adattamento designato elettrico. Tale sistema viene utilizzato in laboratorio Davies 17. Il software LabVIEW e il designato disegno adegua circuito elettrico per la fase di Clifton sono disponibili su richiesta. In conclusione, si descrive un osmometro nanolitri che facilita il controllo sensibile e la manipolazione della temperatura e il tasso di aumento e diminuzione della temperatura (con 0,002 ° C sensibilità), coordinati con un&#39;interfaccia video attraverso una routine LabVIEW per analisi in tempo reale. Questo sistema può eseguire riproducibili tasso di esperimenti controllati che sono important per lo studio della cinetica delle interazioni IBP con ghiaccio. Tali esperimenti possono affrontare diverse a lungo dibattute questioni riguardanti il ​​meccanismo d&#39;azione di IBPs.

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo / modello </td><td> Commenti </td></tr><tr><td> Olio di immersione di tipo B </td><td> Cargille Laboratories </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 soluzione di pulizia </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> Capillare estrattore </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> Tubi di vetro capillari </td><td> Marca GnbH </td><td> 7493 21 </td><td> 75 mm, diametro 1,15 </td></tr><tr><td> Temperaturacontrollore </td><td> Newport, Irvine, California, Stati Uniti </td><td> Modello 3040 </td><td> Modello 3040 </td></tr><tr><td> Microscopio ottico </td><td> Olimpo </td><td> Modello BH2 </td><td></td></tr><tr><td> Obiettivo 10x </td><td> Olimpo </td><td></td><td> S Piano 10, 0.3, 160/0.17 </td></tr><tr><td> Obiettivo 50x </td><td> Nikon </td><td></td><td> CF piano, 50X/0.55 EPI ELWD </td></tr><tr><td> CCD </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tygon tubi </td><td> Saint-Gobain, Parigi, Francia </td><td></td><td> Tygon Formulazione S-50-HL Tubazione </td></tr><tr><td> Siringa di vetro (2 ml) </td><td> Poulten-Graf, Wertheim, Germania </td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-scheda PCI </td><td> National Instruments, Austin, Texas, USA </td><td> 778032-01 </td><td></td></tr><tr><td> Video telaio grabBER IMAQ-PCI-1407 </td><td> National Instruments, Austin, Texas, USA </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW di sistema Software Design </td><td> National Instruments, Austin, Texas, USA </td><td> Versione 8 </td><td></td></tr><tr><td> DivX Author software </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).

Misurazione della dipendenza dal tempo TH Il LabVIEW azionati osmometro nanolitri facilita l&#39;esecuzione di misure accurate attività TH. La costante di velocità di riduzione della temperatura consentita la misurazione della dipendenza dal tempo TH. Il controllo preciso della temperatura abilitato dal osmometro nanolitri era cruciale per questi esperimenti. Il tempo di esposizione di un cristallo di ghiaccio ai IBPs in soluzione è definita come il periodo di tempo dalla formazione del cristallo (la fine del processo di fusione), finché la crescita improvvisa di ghiaccio intorno al cristallo (burst cristallo). Abbiamo trovato che il tempo di esposizione dei cristalli di ghiaccio ai IBPs dipende moltissimo l&#39;attività TH. Brevi periodi di esposizione IBP (alcuni secondi) prodotto una bassa attività TH nell&#39;IBP-GFP soluzione Mp (2,4 pM) (Figura 5). L&#39;attività TH aumentato con il tempo di esposizione IBP fino a raggiungere un plateau a esposizione min 4 IBP. A concentrazioni più elevate IBP, la piastraau è stato raggiunto in tempi più brevi. <img alt="Figura 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> Figura 1. IBPs diagramma schematico che illustra adsorbito al ghiaccio. Adottato con il permesso di 10. <img alt="Figura 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> Figura 2. La fase di raffreddamento. A) collegate a tubazioni sul microscopio. B) Senza la guida superiore. C) Schema di principio. <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. Schermata di interfaccia LabVIEW. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Cl</a>ick qui per ingrandire la figura. <img alt="Figura 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> Figura 4. Stabilità grafico della temperatura. Il regolatore di temperatura è stato impostato per abbassare la temperatura di 0,01 ° C ogni 15 sec. <img alt="Figura 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> Figura 5. Mp attività TH IBP in funzione del tempo di esposizione ai cristalli di ghiaccio IBPs. Ogni punto rappresenta la media di tempo di 3-6 esperimenti.

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

Proteine ​​leganti ghiaccio (IBPs), noto anche come proteine ​​antigelo, inibiscono la crescita di ghiaccio e sono un additivo promettente per l&#39;uso nella crioconservazione dei tessuti. Lo strumento principale utilizzato per indagare IBPs è il osmometro nanolitri. Abbiamo sviluppato una casa progettata fase di raffreddamento montato su un microscopio ottico e controllato utilizzando un custom-built routine di LabVIEW. Il osmometro nanolitri qui descritto manipolato la temperatura del campione in un ultra-minuscole.

LabVIEW a comando nanolitri osmometro Novel per le indagini Protein Ice rilegatura

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 Views.  The Hebrew University of Jerusalem. Proteine ​​leganti ghiaccio (IBPs), noto anche come proteine ​​antigelo, inibiscono la crescita di ghiaccio e sono un additivo promettente per l'uso nella crioconservazione dei tessuti. Lo strumento principale utilizzato per indagare IBPs è il osmometro nanolitri. Abbiamo sviluppato una casa progettata fase di raffreddamento montato su un microscopio ottico e controllato utilizzando un custom-built routine di LabVIEW. Il osmometro nanolitri qui descritto mani...

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.

Passivazione della superficie per singola molecola proteica Studi

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

Ricerca

Chimica

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.

Legame covalente di BMP-2 su superfici utilizzando un auto-assemblato Monolayer Approccio

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.

Determinazione dei piani di legame del ghiaccio delle proteine antigelo mediante affinità del piano di ghiaccio basata sulla fluorescenza

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-strato Interferometria per Cinetica di interazioni proteina-proteina e allosterici effetti Ligand di misura

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) luminescente sonda per il rilevamento della proteina malarica Biomarker istidina 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.

Acqua in emulsioni olio: un nuovo sistema di montaggio idrosolubili proteine ​​clorofilla vincolante con pigmenti idrofobi

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 Based rilegatura e metodo concorrenza di determinare con rapidità interazioni ligando-recettore

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 nativo Gel-eluito Frazione Liquid Entrapment elettroforesi

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.

Indagini su GA (III) Complesso di EOB-DTPA e la sua<sup&gt; 68</sup&gt; Ga marcata radioattivamente analogico

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.

Nuove tecniche per l'osservazione dinamica strutturale dei cristalli liquidi fotoresponsivi

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