Pekin Wu Biz onu veri kullanımı için teşekkür ederim. Bu çalışma, NSF KARİYER Jek için bir ödül tarafından desteklenen

1. Membran Protein katlanması için ~ 50 nm Çapı Küçük Unilamellar veziküller (SUV) hazırlanması <ol><li> 1,2-dimyristoyl-sn-Glycéro-3-Phosphocholin (DMPC) kloroform lipidlerin bir çözüm başına 20 mg flakon miktarlarda depolama için satın alınan ve temiz bir cam şişe içine aliquotted. Azot gazı bir katman yağ oksidasyonunu önlemek için her flakon eklenir ve kapaklar ve parafilm şişeleri ile mühürlenir. Şişeler, kullanılıncaya kadar -20 ° C derin dondurucuda saklanır. </li><li> Kloroform 20 mg lipid bir kısım içeren tek bir flakon, her bir deney için kullanılır. Flakon içeriği, hiçbir solvent kalana kadar 1 saat boyunca azot gazı akışı ile kurutulur. </li><li> Kurutulmuş lipidler, 1 ml 20 mM potasyum fosfat tamponu yeniden süspanse ~ 30 saniye boyunca bir su banyosunda sonikatör kullanarak (pH = 7.3). Bu çözüm, askıya lipid bulanık görünür ve konik bir alt 15-ml plastik bir tüpe aktarılır. Lipid başlangıçta yer alan boş cam şişe 1 ml fosfat tampon diğer kısım eklenir ve sonication tekrarlanır; çözüm daha sonra 15 mL aynı tüpe eklenir. Bu süreç, bir lipid konsantrasyonu 5 mg / ml bu hisse vezikül çözüm ile sonuçlanan tüp nihai hacmi, 4 mL kadar toplam 4 kez tekrarlanır. </li><li> 4 mL hacimdeki lipid vezikül çözüm yerleştirilir sıcak (~ 30 ° C) su banyosu, ve 1 saat süreyle% 50 görev döngüsü ultrasonicator mikrotip kullanarak sonicated. Su banyosunda iki amacı vardır. İlk olarak, sonication süreci nedeniyle çok sıcak hale gelen lipid çözümü engeller. İkincisi, sıcak banyo sulu lipid çözüm sıcaklık sonication sırasında çift katlı faz geçiş sıcaklığı üzerinde kalmasını sağlar; DMPC için, bu geçiş sıcaklığı ~ 23 ° C </li><li> Sonicated çözüm sonikatör ucundan enkaz kaldırmak ve bu filtre edilmiş solüsyon 37 geceleme dengelenmiş filtresi 0.22 mikron şırınga ° C geçirilir </li></ol> 2. İlk Floresan açılması Eğrisi için Numune Hazırlama <ol><li> 20 mM fosfat tamponu (pH 7.3), 10 M üre stok solüsyonu yapılır. Bu büyük miktarda üre çözümünde önemli bir hacim işgal eder, çünkü su katı (ve tersi) eklendi olması esastır. Bu üre çözeltisi temiz, boş şişe çözünenlerin ekleyerek ve amaçlanan nihai hacim su ekleyerek yapılabilir. Bu çözüm, bir su banyosu veya çözünen çözülür karıştırarak sıcak bir plaka üzerinde ısıtılması gerekebilir. Üre higroskopik ve bu nedenle, üre stok solüsyonu gerçek konsantrasyon, kırılma indisi kullanılarak tespit edilmelidir. 1 </li><li> 20 mM fosfat (pH 7.3) stok tampon çözeltisi yapılır. </li><li> Bir stok solüsyonu protein hazırlanan gelişeceğini. Bu stok protein çözümü 8 M üre, 20 mM fosfat tamponu ~ 200 mcM protein konsantrasyonu olmalıdır. </li><li> Floresan çalışmaları için örnekler şu şekilde hazırlanır. Uygun stok protein hacimleri (bölüm 2.3), lipid stok solüsyonu (5 mg / ml yağ, bölüm 1), hisse senedi 10 M üre çözeltisi (bölüm 2.1), ve stok fosfat tamponu (bölüm 2.2) ~ 4 içeren örnekler yapmak için bir araya mcM protein, 1 mg / ml yağ, 1 M artışlarla 0-8 M üre. Her numunenin toplam hacmi 200 mcL. Bir örnek tablo (Tablo 1) listeleri hacimleri 200 mcM stok protein solüsyonu kullanılarak gerekli olduğunu dahildir. </li><li> Boş örnekleri bölüm 2.4 &#39;te açıklandığı gibi yapılır, ancak, protein eklendi değildir. Diğer bileşenlerin konsantrasyonları bölüm 2.4 &#39;te aynı şekilde protein yerine, 8 M üre çözeltisi 4 mcL eklenebilir. Bu boşlukları, saçılma ve protein floresans spektrumları görünen diğer arka plan sinyali çıkarmak için kullanılır. </li><li> 2.4 ve 2.5 bölümleri örnekleri floresans spektrumları ölçümden önce tam katlama ve dengeleme sağlamak için en az 2 saat süreyle 37 ° C&#39;de inkübe izin verilir. </li></ol> 3. Floresan Spectra ölçümü Istikrarlı bir devlet flüorometre kullanılarak her örnek ve boş Triptofan floresan ölçülür. Floresans spektrumları tirozin kalıntılarının uyarma önlemek için 290 nm dalgaboyu ile kaydedilen ve 305 ila 500 nm taranmış olmalıdır. Tipik giriş ve çıkış bant geçiren 3 nm. Dalga boyu arttırma ve entegrasyon süresi, sinyal-gürültü oranı için optimize edilebilir. Dalga boyu arttırma ve entegrasyon zamanı için tipik değerler, 1 nm / adım ve sırasıyla 0.5 sn / adım,. Membran sıcaklığı çift katlı faz geçiş sıcaklığı üzerinde sadece içine genellikle kat sentetik lipidler proteinler. Bu nedenle, DMPC bu durumda, 30 numunenin sıcaklığı sabit tutulur ° C. 160 mcL kapasitesine sahip bir microvolume erimiş silika küvet bu eski kullanılmaktadırperiments. 4. İlk açılım Eğrisi açılması Membran Protein Gibbs Serbest Enerji ve Kestirim Üretimi <ol><li> Xy veri formu Floresans spektrumları Igor Pro, Matlab, Kökeni, veya Excel gibi yazılım yüklenir. </li><li> Sinyal, üre ve lipid vezikül arka plan aşağıdaki yordamı kullanarak çıkarılır olmalıdır: Protein, lipid veziküller ve spesifik konsantrasyon üre varlığını = ham floresans spektrumu Spektrum. Spektrum B lipid veziküller ve aynı üre konsantrasyonu içeren ilgili boş örnek = ham floresans spektrumu bu kadar spektrumda; herhangi bir protein boş örnekleri. Düzeltilmiş Spektrum = Spektrum A - C * spektrum B. C, tipik olarak 1.0 &#39;e eşit bir skaler. Bazı durumlarda, C, çünkü neden saçılma konularda daha az ya da 1.0 &#39;dan büyük veya ~ 305-320 nm bölgeye yakın çıkarma altında. </li><li> Dalga boyu, λ maksimum, maksimum floresans spektrumları her üre konsantrasyonu için düzeltilmiş tablolaştırılmıştır. Tamamen için tipik bir değeri katlanmış proteini ~ 330 nm ise membran protein 350 nm gözler önüne seriliyordu. Tablo dalga boylarında gelişeceğini nüfusu kesir λ max değeri ilişkilendirmek için 1.0 ila 0.0 aralığında λ maksimum değerleri (~ 330 ~ 350 nm) aralığında dönüştürme gelişeceğini fraksiyonu dönüştürülür. Örneğin, 330 nm λ max değeri 0.0 karşılık gelen, 350 nm 1.0 &#39;e karşılık gelir ve 340 nm fraksiyonu açısından 0.5&#39; e karşılık katlanmamış. Bu gelişeceğini protein fraksiyonu sonra üre konsantrasyonu karşı çizilmiştir. Yukarıda belirtildiği gibi, üre konsantrasyonu, refraktif indeks ölçerek deneysel olarak tespit edilmelidir. </li><li> Biz iki devlet bir protein bulunabilir varsayalım: katlanmış veya katlanmamış. Kesir arsa f gelişeceğini, üre konsantrasyonu, C, karşı denkleme uygun olabilir: 2 <img src="/files/ftp_upload/2669/2669eq2.jpg" alt="Denklem 2" /> Katsayısı m denaturant konsantrasyon açısından serbest enerji değişim oranına karşılık gelir ve C m katlanmış nüfus gelişeceğini nüfus eşit olduğu orta noktası üre konsantrasyonu karşılık gelir. Bu değerler R (0.001987 kcal / mol • K) gaz yasası sabittir. Oturan en küçük kareler elde edilen veri analiz yazılımı tarafından uydurma değişkenleri, ve T (303 K) sıcaklık. </li><li> Fit elde edilen katsayılar üre yokluğunda ortaya konma Gibbs serbest enerji (kcal / mol) belirlemek için kullanılır: <img src="/files/ftp_upload/2669/2669eq3.jpg" alt="Denklem 3"/> . </li></ol> 5. Eğri açılması açılması, Gibbs Serbest Enerji Hassas Ölçüm için optimize edilmiştir. <ol><li> Yukarıda açıklanan açılımı eğrisi λ max en büyük değişiklik neden üre konsantrasyonlarının aralığı ortaya koymaktadır. Bu aralık, genellikle küçük ve unfolding çoğunluğu bu küçük aralık içinde gerçekleşir. Açılmadaki serbest enerji tam olarak belirlemek için, optimize edilmiş en küçük kareler montaj için gerekli birçok veri noktaları içeren bir arsa elde etmek için bu bölgede ek örnekler analiz edilir. Daha doğrusu, gözler önüne serilen eğrisi bu önemli bölgenin şeklini ortaya çıkarmak için 0.2 M artışlarla 4 M üre - Örneğin protein, 2 ve 4 M üre arasında gözler önüne serilir, örnekleri 2 içerdiğini yapılmış olabilir. Bu her üre konsantrasyonu boş spektrumları ölçmek için gerekli değildir; her ~ 0.5 M adım boş bir spektrumu ölçmek için yeterli. </li><li> Orijinal gözler önüne serilen eğrisi elde edilen bu arsa elde açılmadaki serbest enerji biraz farklı, ama daha kesin bir değer yansıtır. </li></ol> 6. Temsilcisi Sonuçlar: <table border="1"><tbody><tr><td> Örnek </td><td> son [Üre], M </td><td> stok protein (mcL) </td><td> stok lipid (mcL) </td><td> stok 10 M üre (mcL) </td><td> stok tampon (mcL) </td></tr><tr><td> 1 </td><td> ~ 0.16 </td><td> 4 </td><td> 40 </td><td> 0 </td><td> 156 </td></tr><tr><td> 2 </td><td> 1 </td><td> 4 </td><td> 40 </td><td> 20 </td><td> 136 </td></tr><tr><td> 3 </td><td> 2 </td><td> 4 </td><td> 40 </td><td> 40 </td><td> 116 </td></tr><tr><td> 4 </td><td> 3 </td><td> 4 </td><td> 40 </td><td> 60 </td><td> 96 </td></tr><tr><td> 5 </td><td> 4 </td><td> 4 </td><td> 40 </td><td> 80 </td><td> 76 </td></tr><tr><td> 6 </td><td> 5 </td><td> 4 </td><td> 40 </td><td> 100 </td><td> 56 </td></tr><tr><td> 7 </td><td> 6 </td><td> 4 </td><td> 40 </td><td> 120 </td><td> 36 </td></tr><tr><td> 8 </td><td> 7 </td><td> 4 </td><td> 40 </td><td> 140 </td><td> 16 </td></tr></tbody></table> Tablo 1 floresan örnekleri yapmak için gereken stok çözelti hacmi <img src="/files/ftp_upload/2669/2669fig1.jpg" alt="Şekil 1" /> Şekil 1: tek bir triptofan kalıntı içeren ~ 5 mcM temsilcisi membran proteini Triptofan floresans spektrumları. (A) Ham protein floresans spektrumları (spektrumu 4.2 &#39;den A), (B) boş Ham floresans spektrumları (4.2 spektrum B), (C) 4.2 spektrum düzeltildi. <img src="/files/ftp_upload/2669/2669fig2.jpg" alt="Şekil 2" /> Sayısız üre konsantrasyonları için Şekil 1 Şekil 2 membran protein triptofan floresans spektrumları düzeltildi. <img src="/files/ftp_upload/2669/2669fig3.jpg" alt="Şekil 3" /> Şekil 3 Şekil 2 veri elde 4.4 &#39;denklemine uygun eğri açılması. Gibbs serbest enerjisi, bölüm 4.5 &#39;e göre hesaplanır.

<ol>
	<li>Shirley, B. A., Shirley, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Folding and Stability. , 177-190 (1995).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+and+Analysis+of+Urea+and+Guanidine+Hydrochloride+Denaturation+Curves.">Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves.</a> Methods Enzymol. 131, 266-279 (1986).</li><li>Lau, F. W., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Assessing+the+Stability+of+a+Membrane+Protein.">A Method for Assessing the Stability of a Membrane Protein.</a> Biochemistry. 36, 5884-5892 (1997).</li><li>Burgess, N. K., Dao, T. P., Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Barrel+Proteins+that+Reside+in+the+Escherichia+coli+Outer+Membrane+in+Vivo+Demonstrate+Varied+Folding+Behavior+in+Vitro.">&#946;-Barrel Proteins that Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro.</a> J. Biol. Chem. 283, 26748-26758 (2008).</li><li>Booth, P. J., Curnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+scene+investigation:+membrane+proteins.">Folding scene investigation: membrane proteins.</a> Curr. Opin. Struct. Biol. 19, 8-13 (2009).</li><li>Hong, H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+coupling+of+integral+membrane+protein+stability+to+lipid+bilayer+forces.">Elastic coupling of integral membrane protein stability to lipid bilayer forces.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4065-4070 (2004).</li><li>Sanchez, K. M., Gable, J. E., Schlamadinger, D. E., Kim, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Tryptophan+Microenvironment,+Soluble+Domain,+and+Vesicle+Size+on+the+Thermodynamics+of+Membrane+Protein+Folding:+Lessons+from+the+Transmembrane+Protein+OmpA.">Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA.</a> Biochemistry. 47, 12844-12852 (2008).</li></ol>

Mevcut protokol triptofan artıkları içeren membran ile ilişkili proteinler ve peptitler eğriler açılmadaki nesil açıklamaktadır. Burada, triptofan floresan protein ve sentetik lipid veziküller takılı katlanmış, ya da çözelti içinde gelişeceğini olup olmadığını yansıttığı varsayılır. Gibi iki-devletli katlama ve serbest enerji denaturant konsantrasyonu ile lineer bağımlılığı gibi ek varsayımları, cari rapor yapılır; farklı denklemler bu varsayımların sonucu değiştirilmesi <sup...

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thermodynamics of membrane protein folding measured by fluorescence spectroscopy

Membran protein katlanması ve sağlıkla ilgili temel öneme sahip bir konudur. Membran proteinleri hücrelerin temelini bu proteinlerin her yerde aile katlanır kapsamlı bir çalışma için ihtiyaç bolluğu. Ayrıca, misfolded proteinler ile ilişkili hastalıklar karakterize yeteneğimizi gelişmeler, protein katlanması konusunda önemli deneysel ve teorik çabaları motive etmiş. Bu önemli alanda hızlı bir ilerleme maalesef, membran proteinleri ve katlama mekanizması karmaşıklığı ile ilişkili içsel zorluklar tarafından engellenmektedir. Burada, biz, E. bir temsilci integral membran proteini denaturant yokluğunda, Δ G ° H 2 O açılmadaki Gibbs serbest enerji termodinamik özelliği ölçmek için deneysel bir prosedür anahat coli. Bu protokol, denaturant konsantrasyonunun bir fonksiyonu olarak katlanmış ve gelişeceğini devletlerin denge nüfusu belirlemek için floresans spektroskopisi uygulanması üzerinde duruluyor. Sentetik lipid veziküller yanı sıra veri analizi prosedürü önemli adımlar hazırlanması için deneysel düşünceler vurgulanır. Bu teknik, çok yönlüdür ve denaturant sıcaklık ve pH yanı sıra lipidler ve miseller çeşitli katlanır ortamlar da dahil olmak üzere farklı tipleri ile takip edilebilir. Geçerli protokol kriterler aşağıda tartışılmıştır karşılayan herhangi bir membran veya çözünebilir protein jeneralize olabilir.

Watch this Scientific Journal Video about Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy at JoVE.com

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Bu video makalede, triptofan floresan membran protein katlanması Gibbs serbest enerji elde etmek için deneysel prosedür.

Membran Protein katlanması, Termodinamik Floresans Spektroskopisi ile ölçülür.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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18.2K Görüntüleme.  University of California San Diego - UCSD. Bu video makalede, triptofan floresan membran protein katlanması Gibbs serbest enerji elde etmek için deneysel prosedür.

Video: Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Mikroakışkan damlacık platformları için Floresan algılama yöntemleri

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Biyomühendislik

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Protein Katlama Öğrenmenin mikroakışkan Mikserler

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membran-OMURGA: Membran Proteinleri protein-protein etkileşimleri belirlemek için bir Biyokimyasal Aracı<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Modifiye İki foton mikroskop 3D Orbital Takip: Hücre içi veziküllerin İzlemeye Bir Uygulama

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Ticari Mikroskop Canlı Hücre Zarları Moleküler Difüzyon Kanunu Hızlı Floresans Görüntüleme Gönderen

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin Spektroskopisi için Ekstraselüler Matriks Proteini Elyaf Hazırlama

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Mühendislik kanonik olmayan Amino asitler ve seçici basınç eklenmesi tarafından 'Altın' floresan ve kütle spektrometresi ve floresan göre Protein analizi

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nano-formülasyonları endotoksin tespiti Limulus Amoebocyte Lysate (LAL) deneyleri kullanarak

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Vesiculo 'da otonom vezikül büyümesi için peptid membran öncülerinin sentezini

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

Biyomembran Kuvvet Probu Spektroskopisi ile Moleküler Yay Sabit Analizi

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...