Bu araştırma ISF, NSF ve ERC tarafından desteklenmiştir. Biz Randy Milford, Michael Koren, Doug Shafer, ve Jeremy Dennison sıcaklık kademesi ile teknik yardım kabul etmek istiyorum. Yazılım geliştirme ile Yardımlaşma Veya Chen, Di Xu, Rajesh Sannareddy ve Sumit Bhattachary tarafından sağlanmıştır. Biz Mp IBP protein ve yararlı tartışmalar için ortak Prof Peter L. Davies ve Dr Laurie A. Graham teşekkür etmek istiyorum. Biz de teşekkür laboratuar üyeleri Dr Maya Bar-Dolev, Yangzhong Qin, Dr Yeliz Çelik, Dr Natalya Pertaya, Ortal Mizrahy ve kendi kullanıcı geri bildirimleri için Shlomit Guy.

0. Ön Prosedürler <ol><li> Cam çözüm enjeksiyon için kısımdır. Kılcal çektirmesi (Narishige, Tokyo, Japonya) kullanarak, bir cam kapiller mikro tüp (Marka GMBH, Wertheim, Almanya) ince bir açılış ile keskin bir pipet hazırlayın. Açıklık boyutu temiz su içinde ince köpürme sağlamak için kılcal içinden hava geçirerek doğrulanması gerekir. Kılcal kapalı ise, o zaman bir kenarından kırarak açabilirsiniz. Bu tüp duvarları içeren suya karşı bastırarak ya da hafifçe kazınarak gerçekleştirilebilir. Açılış neredeyse bloke edilir ama yeterince milimetrenin altındaki kabarcık oluşumuna izin açık olduğunu kapiller gibi hazırlayın. </li><li> Bakır disk temizleme. Çift distile su ile yıkayın, daha sonra% 0.1 Mikro-90 sabun (Cole-Parmer, Vernon Hills, Illinois, ABD) 10 dk bakır diskler sonikasyon. Bir izopropanol (teknik) çözeltisi içine diskler tanıtın ve 10 dakika için tekrar sonikasyon. Yüzgeçmüttefiki, filtrelenmiş hava kullanarak diskleri kurutun. Bu temizlik aşamasında deneyler arasındaki IBP kontaminasyonu önlemek için çok önemlidir. </li><li> Çift katmanlı coverglass montaj. A coverglass montaj kapağı cam yüzeyindeki nem yoğuşma olmadan örnek gözlem için izin vermek için hazırlanmıştır. Bu daha sonra bir sıcak tutkal tabancası ile yapıştırılmıştır iki lamelleri arasında Drierite (WA Hammond Drierite, Xenia, Ohio, ABD) parçacık (çapı 2 mm) koyarak elde edildi. Bu konfigürasyon örnek düşük sıcaklıklara soğutulur ve gözlem penceresi üzerinde kuru hava üflemek için ihtiyaç kaldırıldı görünümünü blok olabilir yoğunlaşma engellemiştir. </li></ol> 1. Soğutma Sahne Kurulumu <ol><li> 4 mm iç çaplı Tygon tüp (Saint-Gobain, Paris, Fransa) ile soğutma aşamasında su akışı giriş ve çıkış bağlan, ve bir su pompası, su akışının giriş tüpü bağlanır. </li><li> Soğutma aşaması t girişine 4 mm&#39;lik bir iç çap Tygon tüp bağlankuru hava teslim o. Hava bir in-line Drierite kolon kullanılarak kurutulmuştur. </li><li> Hava ve su pompaları çalıştırın. Soğutma elemanları bir soğutucu olmadan çalıştırmak gerektiğini unutmayın. </li><li> Sıcaklık kontrol, kamera ve LabVIEW rutin açın. </li></ol> 2. Örnek Hazırlanması <ol><li> Disk ile delinen 500 mikron delikleri olan bir 7 mm çapında bakır diskin arka tarafında immersiyon yağı B (Cargille laboratuarlar, Cedar Grove, New Jersey, ABD) bir 3-4 ul damlacığı yerleştirin. </li><li> Aşağı bakacak immersiyon yağı tarafı ile soğutma sahnede bakır diski yerleştirin. </li><li> 2 ml&#39;lik bir cam şırıngaya (Poulten-Graf, Wertheim, Almanya) için diğer uçta bağlanmış bir 0.7 mm iç çaplı Tygon tüp ile bağlantı borusu (küt kenar) bağlanır. </li><li> Önceki kapiler tüp kullanarak, açılış (Ön prosedürler bakın) uygun bir büyüklükte olduğundan emin olmak için kılcal küçük deliğini kontrol edin. </li><li> Yavaşça gla takınss hazırlanan IBP protein örnek tüpü (pH 8 20 mM içinde 2.4 uM Mp IBP-GFP CaCl2 ve 25 mM Tris-HCl, hazırlama ayrıntılar için referans 10) içine kılcal ve kılcal cam 0.1 içerene kadar cam şırıngaya çekme Protein çözeltisi, ul. </li><li> LabVIEW yazılımı ile video kayıt başlayın. </li><li> Soğutma aşamasında bakır disk içinde deliklerin biri içine kılcal cam (protein çözeltisi içeren) keskin kenar yerleştirin. </li><li> (Olympus, Tokyo, Japonya, 10x objektif) mikroskop ile dikkat ederken, dikkatle cam kapiller ucu daldırma yağ tabakası nüfuz ve protein az miktarda (~ 10 nl) sunmak için cam şırınga (çok ince) tuşuna basın 200 mikron damlacık oluşturmak için bir çözüm. </li><li> Çift katmanlı coverglass montaj (Ön Prosedürler bakınız) ile soğutma aşamasında deliği kapatın. </li></ol> 3. TH Etkinlik Ölçümü <ol><li> Önss soğutma düğmesi ve -40 sıcaklığını ayarlamak ° C </li><li> Başlangıçta, çözüm damlacık açık olacaktır. Düşük sıcaklıklarda, tipik aralık içinde -30 ° C ile -35 çözelti dondurulmuştur belirten ° C, damlacık renk değiştirir. Toplu buz erimeye başlayana kadar örnek donduktan hemen sonra, yavaş yavaş sıcaklığını arttırın. Sıcaklığın bir kademeli artış numune tamamen erimesi neden olabilir sıcaklık aşılmaz önlemek için gereklidir. </li><li> Bir 50x objektif geçin ve sıcaklığını ayarlayarak buz erimeye başlar. Bu ayarlama, interaktif, ve son aşamalar, genellikle 0,002 ° C arasında küçük sıcaklık aşamalarını kullanarak gerçekleştirilir Tek bir kristal kalmayana kadar erimeye devam edin. Kristalin nihai boyutu 10 mikron civarında olmalıdır. Erime durdurulmuştur olan en yüksek ısı erime noktası olarak tespit edilir ve daha sonra video analiz aşamada doğru şekilde belirlenir. </li> &lt;li&gt; kristalin erime noktasının altındaki bir santigrat derece arasında birkaç yüzde için sıcaklığına getirin ve 10 dakika gecikme ile bir sıcaklık rampası başlar. Istediğiniz gibi rampalanma oranını ayarlayın. Bu süre zarfında, kristal IBPs maruz kalacağı. </li><li> 10 dakika maruz kalma süresi tamamlandıktan sonra, sıcaklık LabVIEW rutin otomatik olarak kontrol altında azalacaktır. </li><li> Sıcaklık azaldıkça olarak kristal şekli dikkate alınmalıdır. Bir noktada, buz kristali ani patlama görülebilir. Bu gerçekleştiği sıcaklık kristal patlama sıcaklığı olarak not edilir. </li><li> Doğru erime noktası ve patlama sıcaklığı belirlemek için video analiz kullanın. İlk olarak, görüntü analizleri kullanılarak, kesin erime noktası bulabilirsiniz. Erime bittikten hangi yüksek sıcaklık erime noktası olmaya kararlı olduğunu hatırlayın. Bir elektronik tablo programı bu erime noktası belgeleyin. Sonra, hassas kristal patlama sıcaklığı belirlemek ve bu değeri belgelemekgibi. Erime noktası ve donma noktası, veya kristal patlama sıcaklık arasındaki fark IBP çözeltisinin termal histerezis faaliyettir. </li></ol> 4. Zamana bağlı TH Aktivitenin Ölçümü <ol><li> Buzun tek bir kristali hazırlamak için Bölüm 3.1-3.3 de tarif edilen protokol takip edin. </li><li> Kristal oluşumunu sonra, istediğiniz gibi rampa gecikme süresini ayarlamak ve rampa açın. </li><li> Sıcaklığı otomatik kez rampa gecikme süresi dolduktan (operatörlerinin ihtiyaçlarına göre) sabit bir oranda azalacaktır. </li><li> Kristal çoğuşma oluştuğu sıcaklık belge. Pozlama süresi (kristal oluşumu ve kristal patlama arasındaki zaman) hesaplayın. </li><li> Çeşitli gecikme zamanları için deney tekrarlayın ve TH aktivitesi zaman bağımlılığı değerlendirmek için maruz kalma süresinin bir fonksiyonu olarak TH aktivitesi çizmek. </li></ol>

<ol>
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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>

Çıkar çatışması ilan etti.

Bu çalışma olağanüstü ısı kontrolü ile TH aktivitesinin doğru ölçümleri mümkün kılar bir bilgisayar kontrollü nanolitre Ozmometre çalışmasını gösterir. Herhangi bir sıcaklık duyarlı sisteminde, istenmeyen sıcaklık değişimleri kaçınılmalıdır. Burada sunulan cihaz içinde sıcaklık gradyanları önlemek için, test çözeltisi damla bakır disk soğuma evresi (adım 2.7) içinde bir deliğin merkezi olarak konumlandırılmış olmalıdır. Ayrıca, tek bir kristal damlacık merkezinde ziyade kenarlarına yakın olmalıdır (çoğu durumda, bu kendiliğinden gerçekleşir). Açıklandığı zaman bağımlılığı soğutma hızı TH okumalarını etkiler olabileceğini gösterir. Böylece, kristal soğutma önce çözelti maruz kaldığı süreyi bir rapor, hem de soğutma hızı dahil olmak üzere göstermektedir. Biz genellikle 0.01 sıcaklık aşağı Ramping önce 10 dakika bekledi ° C her 4 sn adımlar. LabVIEW kontrollü işbirliğioling aşama microfluidic cihazlar termal olarak manipüle edilebilir, üzerinde bir ters mikroskop ile kullanım için adapte edilmiştir. Bu sistem eGFP, 9, 10, 16 ile etiketlendi buz kristalleri ve IBPs içeren çözelti değiş tokuş deneylerin yapılması kolaylaştırır. LabVIEW kontrollü sistem belirli bir uyum elektrik devresi yoluyla 3.040 sıcaklık kontrol bağlayarak bir Clifton aşama için adapte edilebilir. Böyle bir sistem, Davies laboratuvar 17 işletilmektedir. LabVIEW yazılım ve Clifton aşaması için belirlenen adapte elektrik devresi tasarımı istek üzerine mevcuttur. Sonuç olarak, hassas kontrol ve manipülasyon sıcaklık ve gerçek zamanlı analiz için LabVIEW rutin yoluyla bir video arayüzü ile koordineli sıcaklık artış ve azalış oranı (0.002 ° C hassasiyet ile), kolaylaştıran bir nanolitre Osmometer açıklar. Bu sistem importan vardır tekrarlanabilir hızı kontrollü deneyler gerçekleştirebilirsinizbuz ile IBP etkileşimlerinin kinetik araştırmak için t. Bu tür deneyler IBPs etki mekanizması çevreleyen birkaç uzun zamandır tartışılan sorunları ele alabilir.

<table border="1"><tbody><tr><td> Isim </td><td> Şirket </td><td> Katalog Numarası / model </td><td> Yorumlar </td></tr><tr><td> Daldırma yağı Tip B </td><td> Cargille Laboratuvarlar </td><td> 16484 </td><td></td></tr><tr><td> Drierite </td><td> WA Hammond Drierite </td><td> 043.063 2270g </td><td></td></tr><tr><td> Mikro 90 temizleme solüsyonu </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> Çektirme Kapiller </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> Cam kılcal tüpler </td><td> Marka GNBH </td><td> 7493 21 </td><td> Uzun 75 mm, 1.15 çapında </td></tr><tr><td> Sıcaklıkkontrolör </td><td> Newport, Irvine, California, Amerika Birleşik Devletleri </td><td> Model 3040 </td><td> Model 3040 </td></tr><tr><td> Işık mikroskobu </td><td> Olimpos </td><td> Model BH2 </td><td></td></tr><tr><td> 10x objektif </td><td> Olimpos </td><td></td><td> S Planı 10, 0.3, 160/0.17 </td></tr><tr><td> 50x objektif </td><td> Nikon </td><td></td><td> CF planı, 50X/0.55 EPI ELWD </td></tr><tr><td> CCD Kamera </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tygon tüpü </td><td> Saint-Gobain, Paris, Fransa </td><td></td><td> Tygon Formülasyon S-50-HL Boru </td></tr><tr><td> Cam şırınga (2 mi) </td><td> Poulten-Graf, Wertheim, Almanya </td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-PCI kart </td><td> Ulusal aletleri, Austin, Texas, ABD </td><td> 778032-01 </td><td></td></tr><tr><td> Video frame gragrafik çubuklarıyla bBER IMAQ-PCI-1407 </td><td> Ulusal aletleri, Austin, Texas, ABD </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW Sistem Tasarım Yazılımı </td><td> Ulusal aletleri, Austin, Texas, ABD </td><td> Sürüm 8 </td><td></td></tr><tr><td> DivX Author programı </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).

TH zaman bağımlılık ölçümü LabVIEW çalışan nanolitre Osmometer doğru TH aktivitesi ölçümleri performansı kolaylaştırır. Sabit sıcaklık azalma oranı TH zaman bağımlılık ölçümü izin. Nanolitre Osmometer tarafından etkinleştirilmiş hassas sıcaklık kontrolü bu deneyler için çok önemliydi. Çözelti içinde IBPs, bir buz kristalinin maruz kalma süresi kristali (kristal burst) etrafında buz ani büyüme kadar kristal oluşumu gelen zaman aralığı (erime işleminin sonunda) olarak tanımlanır. Biz IBPs için buz kristallerinin pozlama süresi önemlisi TH aktivitesini etkilediği belirlenmiştir. IBP pozlama (birkaç saniye) kısa dönemler Mp IBP-GFP çözeltisi (2.4 uM) (Şekil 5), düşük TH aktivitesi üretti. Bu 4 dk IBP pozlama bir platoya ulaştı kadar TH aktivitesi IBP pozlama süresi artmıştır. Yüksek IBP konsantrasyonlarda, plakaau kısa zamanlarda ulaşılmıştır. <img alt="Şekil 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> Şekil 1. Şematik gösteren IBPs buz adsorbe. 10 izni ile benimsendi. <img alt="Şekil 2," fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> Şekil 2. Soğuma evresi. A) mikroskop tüpler bağlandı. B) Üst kurşun olmadan. C) şematik diyagramı. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="Şekil 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> Şekil 3. LabVIEW arayüzü ekran görüntüsü. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Cl</a>büyük bir rakam görmek için buraya Ick. <img alt="Şekil 4," fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> Şekil 4. Sıcaklık stabilitesi grafiği. Sıcaklık kontrol sıcaklığı 0.01 ° C, 15 sn düşürmek için kurulmuştur. <img alt="Şekil 5," fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> Şekil 5. IBPs buz kristali kalma süresinin bir fonksiyonu olarak Mp IBP TH aktivitesi. Her zaman noktasında 3-6 deneylerin ortalamasıdır.

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

Ayrıca antifriz proteinleri olarak bilinen buz bağlayıcı protein (IBPs), buz büyümesini engelleyen ve doku dondurma kullanım için umut verici bir katkı maddesi vardır. IBPs araştırmak için kullanılan başlıca araç nanolitre Osmometer olduğunu. Biz bir optik mikroskop üzerine monte edilmiş bir ev tasarlanmış soğutma aşamasında geliştirilen ve özel olarak geliştirilmiş bir LabVIEW rutin kullanılarak kontrol. Nanolitre Osmometer ultra-hassas bir şekilde numune sıcaklık manipüle burada açıklanan.

Buz Bağlayıcı Protein İncelemelerde LabVIEW çalıştırılan Roman nanolitre Ozmometre

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 Görüntüleme.  The Hebrew University of Jerusalem. Ayrıca antifriz proteinleri olarak bilinen buz bağlayıcı protein (IBPs), buz büyümesini engelleyen ve doku dondurma kullanım için umut verici bir katkı maddesi vardır. IBPs araştırmak için kullanılan başlıca araç nanolitre Osmometer olduğunu. Biz bir optik mikroskop üzerine monte edilmiş bir ev tasarlanmış soğutma aşamasında geliştirilen ve özel olarak geliştirilmiş bir LabVIEW rutin kullanılarak kontrol. Nanolitre Osmometer ultra-hassas bir ...

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.

Tek molekül Protein Araştırmaları Yüzey Pasifleme

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

Kimya

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.

Kovalent Öz-monte tabakalı Yaklaşım Kullanarak Yüzeyler üzerinde BMP-2 bağlanması

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.

Floresan Bazlı Buz Düzlemi Benzeşimi ile Antifriz Proteinlerinin Buz Bağlayıcı Düzlemlerinin Belirlenmesi

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.

Protein-protein etkileşimleri ve Allosterik Ligand Etkileri Kinetiği Ölçüm Bio-katman Interferometri

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.

Malarya Protein Biyomarker Histidin Zengin Protein-II Tespiti için İridyum (III) Lüminesans Probe

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.

Petrol Emülsiyonlar Su: Hidrofobik Pigmentler ile Suda çözünür Klorofil bağlayıcı Proteinler Montaj için Yeni Bir Sistem

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.

Bir ELISA Tabanlı Bağlama ve Rekabet Yöntemi Hızla Ligand-reseptör Etkileşimleri belirleme

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 - net yerli jel-elüt sıvı kısmı Tuzak Elektroforez

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.

EOB-DTPA ve Ga (III) Complex Araştırmalar Onun<sup&gt; 68</sup&gt; Ga Radyoaktif Analog

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.

Yapısal Photoresponsive sıvı kristaller dinamikleri gözlemlemek için yeni teknikleri

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