This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. Lipid Preparasyonu <ol><li> Saydam bir cam tüp içine istenilen miktarda lipit bölmeyin. Kullanım E. E. coli kutuplu lipit özü, ama en lipit bileşimler kullanılabilir. Lipidler toz biçiminde ise, 20 mg / ml &#39;lik bir konsantrasyona kadar kloroform bunları yeniden süspanse edin. Bütün solvent buharlaşana kadar N2 gazı altında oda sıcaklığında lipitler kurutun. </li><li> Pentan lipitleri tekrar ve kloroform kalan izleri kaldırmak için yeniden gaz N 2 akışı kurutun. Kuru bütün solvent çekinceye kadar. Kurutma sırasında, lipid altındaki bir biçim verme yoğun kütle yerine borunun alt üçte birini örtmekte olan eşbiçimli bir film dağıtılır ve böylece tüp hafifçe döndürün. </li><li> Sulandırma tamponu içeren bir deterjan ekleme (300 mM KCI, 50 mM sitrik asit, 25 mM K 2 HPO 4, pH 4.5, 35 mM CHAPS) lipidler eritin. Protein CHAPS&#39;tan kötü hoşgörülü ise bu adım için diğer hafif deterjanlar kullanın. </li><li> ResuspenBir su banyosu sonikatör kullanarak netlik lipidler d. Kısa (30 sn-1 dk) ile kısa (30 sn-1 dk) bakliyat maksimum güçte su banyosunda sonikatör merkezinde tüp daldırın duraklar. Çözelti berrak hale gelmelidir. Not: açıklık son derece elde kullanılan spesifik yağ bileşimine bağlıdır. Da nominal olarak denk lipidler arasında bu adımda bazı partiden partiye değişkenlik, aynı zamanda mümkündür. Kalıntı bulanıklık santrifüjleme ile çıkarılabilir süspansiyonda büyük parçacıklar tarafından ışık saçılımı kaynaklanmaktadır. </li><li> 20-60 dakika boyunca oda sıcaklığında yeniden süspansiyon haline lipitler inkübe edin. </li></ol> 2. Proteoliposome Formasyonu Not: birkaç strateji lipozomlara deterjan çözülmüş protein eklemek için kullanılabilir. CLC-EC1 için diyaliz iyi çalışır ve bu nedenle tercih 6,9,10 bir yöntemdir. <ol><li> Saflaştırılmış protein ekleyin istenen protein-to-lipid ug o ifade (P / L) oranı (<html> F proteinine mg / lipid). P / L oranının seçimi deney amacına bağlıdır. <ol><li> Amaç, ilgi konusu proteinin aktivitesini değerlendirmek yüksek P tekrar oluşturmak için, her lipozom proteininin birden fazla kopyasını içerdiğinden / L ile, sinyali en üst düzeye çıkarmak için. Her bir kesecik ortalama 1 protein ihtiva edecek şekilde aktivitesi ve / veya proteinin üniter nakil oranını ölçmek için, düşük P / L&#39;nin de bir Poisson seyreltme rejiminde sulandırın. Her rejimi kullanmak için ne zaman bir daha derinlemesine analiz için Adım 7 ve Tartışma bakın. </li></ol></li><li> Farklı tedavi rejimleri için uygun P / L değerlerini belirlemek için, protein konsantrasyonu 8,11-13 bir fonksiyonu olarak aktivitesi tam bir titrasyon yapmak. Bununla birlikte, aktif biriminin moleküler ağırlığı (MW kDa olarak belirtilmiştir) için kullanılan herhangi bir protein için, aşağıdaki P / L değerleri ilk guesstimates olarak kullanılabilir bilinmektedir: pg &quot;/&gt; <img alt="Denklem 2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> Önceden ıslatılmış diyaliz tüp veya bir kaset protein ve lipit karışımı yükleyin. 500-1,000x lipit yeniden oluşturma tampon hacmi ihtiva eden bir beher içinde diyaliz cihazı yerleştirin (yani, yağ yeniden süspanse edilmesi için 1 ml 1 L tamponu) hafif karıştırma altında. <ol><li> Değişim tampon aralıkları&gt; 3 saatte 3-4 kez. Her çözelti, değişiminde, kaset / boru ve damıtılmış su ile beher yıkayın ve taze tampon maddesi ile yeniden oluşturma beher doldurun. Çözelti değişikliği için 1-2 O / N aralıklarını ekleyin. çözüm değişiklikleri tam sayısı ve süresi kullanılan tam lipid ve deterjanlar bağlıdır. </li></ol></li><li> Son adım tamamlandıktan sonra, diyaliz tekneden lipozomlar kaldırmak tüpler onları bölmeyin ve flaş -80 ° C&#39;de sıvı N 2 veya dondurularak onları dondurmak. </li></ol> 3. Kayıt Ayarı &lt;p class = &quot;jove_content&quot;&gt; NOT: Kayıt set-up (Şekil 2A) iki odadan (düz tabanlı silindir, ~ 3-4 ml hacimli), bir Cl oluşur - elektrot, bir analog bir pH metre (aşağıya bakınız) dijital elektrik çıkışı, bir sayısallaştırıcı ve uygun bir satın alma yazılımı ile bilgisayar veya. <ol><li> PH metre elektrodu, bilgisayara bağlı olan sayısallaştırıcıya pHmetrenin çıkışını - Cl bağlayın. Bir odasında referans elektrot ve diğer (Şekil 2B) içinde recoding teli yerleştirin. NOT: ΔV Cal pozitif (Adım 6.4 ve 7.2) ise tellerin kutuplarının doğru. </li><li> Kayıt odasına (Şekil 1B) ve bir karıştırma çubuğu olan bir karıştırma plakası üzerinde odaları yerleştirin. Karıştırma çubuğu elektrot temas etmediğinden emin olun. </li><li> Bir agar köprü (Şekil 2B) (100 mM KCI ve% 2 agar) ile ölçme cihazı. 100 mM KCl Agar köprüleri saklayın. </li&gt; </ol> Cl 4. hazırlanması - Elektrot <ol><li> Eski ve -possibly- çalışmayan pH elektrodu alın. Tamamen gümüş teller ortaya çıkarmak için cam kaplama kırın. </li><li> Dikkatlice neşter bıçak kullanarak gümüş teller kaplama çıkarın. H2, O ve EtOH bol miktarda kullanarak telleri temizleyin. </li><li> Teller kadar FeCl3 (ya da ağartıcı) bir doymuş çözeltisi içinde yer AgCİ muntazam bir koyu kaplama ile kaplanmıştır. </li></ol> Tek tabakalı veziküller 5. N- <ol><li> deneyler önceki gece, dış tamponu, 15 ml (1 mM KCI, SO 4, 25 mM sitrik asit, pH 4.5, 150 mM K 2), hafif sallama ile (kımıldamam) içinde Sephadex G-50 taneleri 1 g kabarma Kullanmadan önce oda sıcaklığında en az 3 saat. Her bir deney, şişmiş boncuk ~ 3 ml gerektirir. En fazla birkaç gün 4 ° C&#39;de şişmiş boncuk tutun. </li><li> Lipozomlar oda sıcaklığında çözülmesi ederken, her sütun ~ 3 m dökmekşişmiş G-50 taneleri l. Oda sıcaklığında yerçekimi akışı ile onları kuru edelim; Bu genellikle 1-2 dakika sürer. </li><li> 0.4 m, teflon kesme kullanarak Mini-ekstruderinden proteoliposomes 11 kes ekstrude tek lamelli kesecikler hazırlanır. </li><li> Plastik yuvarlak dipli bir tüp içinde sütun yerleştirin. Fazla çözüm kaldırmak için sütunları Spin. Bir klinik santrifüj cihazında 1400 x g&#39;de 20-30 saniye santrifüjleyin. </li><li> 13 x 100 mm cam tüpe akış yoluyla yer sütun atın ve kolona ekstrüde veziküller 100 ul ilave edin. Bir klinik santrifüj cihazında 500 xg&#39;de 1 dakika için sütun dönerler. </li><li> Aşama 6.5 kayıt odasına eklenecek akış yoluyla ve ~ 200 ul toplayın. </li></ol> 6. akış ölçümü <ol><li> Referans (toprak) bölmesi içinde 100 mM KCl 2 ml ve dış tampon 1.8 ml yerleştirin (1 mM KCI, 150 mM K 2 SO 4, 25 mM sitrik asit, pH 4.5), kayıt odasına. İç arasındaki hafif ozmotik dengesizlikve dış çözümler etkilemez </li><li> Satın alma programını başlatın. Bu birkaç dakika sürebilir, temel muvazene edelim. </li><li> Sinyal istikrarlı bir temel (lipozomlar hazır ve sütunlar kurumaya) ulaştığında, (Şekil 3A) kaydetmeye başlayın. </li><li> Sistem (Şekil 2A) kalibre edilmesi için bir 10 mM KCI çözeltisi 15 ul ekle. </li><li> Lipozomlar ekleyin. Proteoliposomes (Şekil 3A) - küçük atlama nedeniyle dış Cl eksik çıkarılması için görünür olabilir. Bazal stabilize kadar bekleyin. </li><li> Valinomycin ekleyin (1 mg EtOH / ml 1 ul) akış (Şekil 3A) gerçekleştirmek için kullanılır. </li><li> Akış zamanı kursu bu yaylalar kadar (Şekil 3A) çalıştıralım. </li><li> Tüm lipozomlar (Şekil 3A), çözünmesi için dış tamponu içinde hazırlanmış 1.5 M β-oktilglükozit (β-RG) 40 ul ekle. Kaydı bitirmek. </li></ol> 7. Veri Analysis <ol><li> Bir ASCII veya metin biçiminde gelen izleme dosyası dışa ve seçim analizi programına ithal. </li><li> Aşağıdaki noktaları (Şekil 3A) Gerilim ölçün: Kaydın başlangıcında (V 0); Ayarlama sinyali (V cal) ilave edildikten sonra; Lipozomlar (V lipo) ilave edildikten sonra; Efflux (V kanat) sonunda; Deterjan (V tot) ilave edildikten sonra; Temel gerilimler böylece V 0 = 0. </li><li> Deneysel değerini belirlemek için, Nernst-Plank denklemi kullanın <img alt="Denklem 3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> ölçülerek <img alt="Denklem 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> Nerede Cilt Cal ul kalibrasyon nabız hacmi deneyi ifade başında odası hacmi, 1.800 olduğunu1; - kalibrasyon nabız ve mM dış tampon konsantrasyonları ve ΔV cal kalibrasyon darbesi sonrasında ölçülen mV atlama l [CI] cal / Cl vardır. NOT: α Bu ampirik belirlenmesi üç amaca hizmet eder: sistem düzgün yanıt olmasını sağlar, deneyler arasındaki enstrümanın tepki tutarlılık ölçüsü sunuyor yanı sıra hiçbir hata çözüm yapımında yapılmış olduğunu kontrol etmek. </li><li> Aşağıdaki gibi her adımdan sonra Cl konsantrasyonları hesaplayın: <img alt="Denklem 5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="Denklem 6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="Denklem 7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> faktör 3/400 2000 ul nihai hacim içinde 15 ul kalibrasyon darbe seyrelmesi gelir. </li><li> CalculHer adımda, fin ΔCl lipo / / tot de [Cl] değişiklikleri yedik. </li><li> Ile Cl (t) v (t) dönüştürme <img alt="Denklem 8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> Doğrudan kritik noktalarda [CI] değerlerini belirlemek ve bir iç kontrol olarak hesaplanan değerler bunları karşılaştırmak. </li><li> Iz Normale Cl rel = 0 lipo ve Cl rel tot = 1 böylece <img alt="Denklem 9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> Aşağıdaki denkleme akış süresi kursu Fit: <img alt="Denklem 10" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> Deneysel olarak Cl ölçülerek belirlenir sızıntısı - - L Cl oranı olduğu durumda, aynı koşullar ve τ hazırlanan proteinsiz lipozomlardan akışını işleminin zaman sabitidir. </li><li> V, ilk hız hesaplayın: Nerede ΔCl fin milimolar ifade ve V ul odasının hacmi olduğunu. </li><li> Taşıma hızı / iletkenlik hesaplayın <img alt="Denklem 12" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> P P / L olduğu durumda, molekül ağırlığı aktif kompleks molekül ağırlığı kDa olarak ifade edilen </li></ol>

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G., del M&#225;rmol, ., J, R., MacKinnon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Human+K2P+TRAAK,+a+Lipid-+and+Mechano-Sensitive+K++Ion+Channel.">Crystal Structure of the Human K2P TRAAK, a Lipid- and Mechano-Sensitive K+ Ion Channel.</a> Science. 335, 436-441 (2012).</li><li>Jayaram, H., Robertson, J. L., Wu, F., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+slow+CLC+Cl-/H++antiporter+from+a+cyanobacterium.">Structure of a slow CLC Cl-/H+ antiporter from a cyanobacterium.</a> Biochemistry. 50, 788-794 (2011).</li></ol>

The authors declare no competing financial interests.

Saflaştırılmış anyon-seçici kanallar veya lipozomlar içinde yeniden taşıyıcıları aracılığı ile ulaşım - Biz detaylı Cl ölçmek için protokol tanımlanmıştır. CLC-EC1 eşanjöre - kullanılan örnek Prokaryotik H + / CI oldu. Söz konusu iyon için uygun biri ile AgCİ elektrot: Ancak, bu yöntemin hali hazırda ligandlar 12,13,15, gerilim 11,12, veya Ag değiştirerek farklı anyonik seçicilik 15,16 spor geçitleri kanalları çal...

Son iki aşırı ifade eden ve zar nakil proteinleri saflaştırmak için yeteneği önemli ölçüde artmıştır yıllarda: iyon kanalları, birincil ve ikincil taşıyıcıları rutin heterolog sentezleme sistemleri ile olduğu gibi, doğal kaynaklardan saflaştırılır. Bu proteinlerin istikrar, ifade izlemek geliştirmek ve çıkarma kolaylaştırmak ve geliştirmek için yeni yaklaşımlar sürekli 1-5 geliştirilmektedir. Bu teknolojik devrimler da, kendi işlevinin yapısal üsleri anlayışımızı gelişmiş, membran proteinlerinin atom düzeyinde yapısal bilgi patlamasını tetikleyen vesile olmuştur. Buna karşılık, bizim yetenek bazı durumlarda yüksek çözünürlüklü yapısal bilgiler, böylece kantitatif yapı-tabanlı tahminler sınamak için yeteneğini sınırlayan, nitel verilerle fonksiyonel eşlik böylece, aynı oranda artmadı saflaştırılmış proteinlerin fonksiyonel özelliklerini incelemek için. Bu nedenle, gelişme içerisindenicel ve genellenebilir fonksiyonel testlerin t membran proteini fonksiyonu mekanik temellerinin aydınlatılması yönünde önemli bir adımdır. Kanal, taşıyıcı - Burada kantitatif saflaştınldı ve yeniden Cl anahtar işlevsel özelliklerini belirlemek için kullanılabilecek bir akış deneyi tarif eder. tahlil altında yatan ilkeler taşıma sistemlerinin çeşitli yanı sıra sigara iyon-taşıma proteinlerine jeneralize olabilir. Kanal / büyük Cl mevcudiyetinde taşıyıcıları - - Lipozomlar saflaştırılmış Cı ile yeniden degrade (Şekil 1A, B). Cı - akış bizim durumumuzda, karşı-iyon akışının izin vermek için bir iyonofor ilave edilerek başlatılır K + iyonoforu Valinomycin, Cl tarafından kurulan gerilim şant olan - gradyeni ve K denge potansiyeline ilk membran potansiyeli ayarlanmış + 6,7. T olmadanBir transmembran potansiyeli nesil tarafından engellenir gibi akış oluşur, - o anlamlı net Cl iyonofor&#39;un. - efflux, f 0, aktif protein içermeyen lipozomlar fraksiyonu τ, Cl zaman sabiti: Veri kantitatif iki ölçülebilir parametreler (Şekil 1C) ile tarif edilir. Τ ve f 0 yekpare Cı kaynaktan - taşıma hızı, aktif protein fraksiyonu ve aktif kompleks moleküler kütlesi 8 elde edilebilir. Bilinen yapısı ve fonksiyonu değiştirici - tekniği CLC-EC1, iyi karakterize CLC-tipi H + / Cl ile yeniden proteoliposomes kullanarak burada gösterilmektedir. Bu tahlil kolaylıkla aktivite gerilimi ve / veya ligandların varlığına bağlıdır farklı iyonik seçicilik veya kanallar veya nakliyecilere genelleştirilmiş. Bundan başka, bu deney, küçük moleküller, doğrudan yeniden protein etkileyip etkilemediğini belirlemek için kullanılabilir,Bu bileşiklerin ve nasıl membran bileşimi veya lipid-modifiye reaktifler yeniden kanalların ve taşıyıcıların fonksiyonunu etkileyebilir etkilerini ölçmek için.

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a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

Değiştirici, lipozomlar içinde yeniden - saflaştırılmış CLC-EC1, bir prokaryotik CLC-tipi H + / Cl aracılığı taşıma - Biz detaylı ve sağlam Cl ölçmek için protokol açıklar. Deneyin şematik bir temsili Şekil 3 &#39;de gösterilmiştir, saflaştırılmış CLC-EC1 ve yüksek iç Cl ihtiva ile yeniden Proteoliposomes -., Düşük Cl ihtiva eden bir banyo çözeltisine daldırıldığı -. Bu koşullar, net Cl altında -</sup...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Bir Proteoliposome Tabanlı effluks Deney Cl Tek-molekül Properties belirleme<sup&gt; -</sup&gt; Kanallar ve Nakliyeciler

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.7K Görüntüleme.  Weill Cornell Medical College. Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Video: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Çift katlı Elastik Özellikleri İzleme Değişiklikler için Tek Molekül Yöntemleri

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Biyoloji

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Floresan Mikroskop ile görselleştirme Tek molekül DNA replikasyonu

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

Enzimlerin Klonlanması DNA Doğrudan Gözlem Tek bir molekülü DNA Testi Esneme

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Tek Molekül Görüntüleme Nükleer Ulaştırma

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRK Kanal Modülatörler belirlenmesi için Floresan Tarama Testi

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Tek Molekül FRET DNA döngü oluşturma incelenmesi

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Laktat dehidrogenaz haciz tahlil — Toplu Autophagic haciz etkinliğini memeli hücreleri belirlemek için basit ve güvenilir Yöntem

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Siteye Özgü DNA Bölünmesi için Paralel Yüksek İş Kinetik Test

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...