Bu araştırma SH Deutsche Forschungsgemeinschaft GraKo1121, Hu1011/2-1 ve SFB940 hibeler ile desteklenmiştir

Not: protokolde belirtilen tamponlar ile ilgili detaylı bilgiler Tablo 1&#39;de kullanılabilir. 1.. Formaldehit Yaşayan hücreler içinde Çapraz bağlama ile protein-protein etkileşimleri Fixation <ol><li> Büyüme ortamı, stok çözümleri ve tampon P1 hazırlamak başlamak için <ol><li> 500 ml ortam hazırlayın: orta yem zar proteini ve av proteinleri arasındaki etkileşimi destek koşulları sağlamalıdır. Buna ek olarak, bir deney bir etkileşim (çapraz bağlanması olmayan, örneğin) beklenen koşullar altında gerçekleştirilmelidir. Not: stresli olan ve olmayan stresli bakterilerde protein-protein etkileşimleri karşılaştırın. Bu nedenle, bir 2 L Erlenmeyer şişesi içinde stres yaratıcı bir madde (örneğin, 0.5 M NaCI) ile tamamlamak 500 ml Luria Bertani (LB) ortamı (Tablo 1) hazırlanması. Kontrolü için, 0.17 M NaCl (pH 7.0) normal LB ortamını kullanın. </li><li> Tris-tamponu hazırlayın (20 mM Tris-HCl, pH 8.0) ve 0.1 M etilendiamin tetraasetik asit (EDTA), pH 8.0 stok çözeltisi (Tablo 1). </li><li> Tampon P1 hazırlayın. Tris-tampon maddesi içinde 0.5 M bir son konsantrasyona kadar sakroz çözülür. Süzme ile sterilize tampon P1 ve 4 ° C&#39;de saklayın </li></ol></li><li> Bir vektör bir C-terminal Strep-eklentisi füzyonu ile yem zar proteini sentezleyen hücrelerin büyümesine. Yeterli bir süre için yem zar proteinlerinin ifadesini teşvik eder. Not: Bu 500 ml ortam için 250 ul 1 M izopropil-β-D-tiogalaktopiranosid (IPTG) eklenmesi ile erken-log fazında (A 600 = 0.3) de (nihai konsantrasyon: 0.5 mM), bakteriyel yem zar proteinlerinin sentezlenmesini uyaran . Yeterli ifadesine olanak tanımak için, geç log fazında (A 600 = 1.2) kadar bakteri inkübe edin. </li><li> 300 ml&#39;lik bir minimum hacimde her iki kültür santrifüj bardak hazırla ve buz üzerine yerleştirin. </li><li> Formaldehit çapraz bağlanmasını gerçekleştirmek. CAUDOĞRULANAMADI: Formaldehit son derece toksik Biz güvenlik davlumbaz altında çalışmanızı öneririz.. <ol><li> Bir güvenlik davlumbaz altında kültür damarı aktarın. (- X) ve çapraz bağlama formaldehid (+ X) da dahil olmak üzere bir iki numune, çapraz-bağlama atlama içine her bir örnek ayrıldı. % 0.6 &#39;lik bir son konsantrasyon elde etmek üzere 250 ml kültüre 4 ml% 37 formaldehit çözeltisi ekleyin. </li></ol></li><li> Geri kültür damarları transferi ve daha önce olduğu gibi 20 dakika daha hücreler büyür. </li><li> Bir güvenlik davlumbaz altında santrifüj tüplerine içine kültürleri doldurun. 30 dakika boyunca 3000 x g&#39;de santrifüj ile hücreler toplayın. </li><li> Dikkatli bir güvenlik davlumbaz altında yukarı pipetleme süpernatant atın. Toksik formaldehit içeren süpernatant toplayın ve uygun şekilde atın. </li><li> Membran proteini hazırlanması sırasında en iyi verimi ulaşmak için, ilk olarak spheroplastlar hazırlar. Burada, Gram-negatif bakterilerin sferoplast oluşumu için bir protokol sağlar: <ol><li> Prepare tampon P2 (Tablo 1) liyofilize toz. Yavaşça, 1.5 ml bir tüp içinde, 0.1 M EDTA, pH 8 içinde 2 mg lizozim çözülür. Her deney günü için taze tampon P2 hazırlar. </li><li> Proteaz inhibitörü (tampon P3) ile Tris-tamponu hazırlayın. Tris-tampon maddesi içinde 10 ml&#39;lik, 10 mM&#39;lik bir son konsantrasyona değin 0.1 ml 1 M fenilmetilsülfonilflüorür (PMSF) (Tablo 1) ekleyin. Proteaz inhibitörü olarak PMSF düzgün çalışmasını sağlamak için, hemen tampon kullanın. </li><li> Proteaz inhibitörü ve bir 15 ml konik tüp transfer çözeltisi ile 10 ml Tris-tampon maddesi içinde yeniden süspanse hücre topakları. </li><li> 1 ml tampon P2 ilave edin ve 30 dakika boyunca buz üzerinde inkübe edilir. </li><li> 30 dakika boyunca 3000 x g&#39;de santrifüj ile spheroplastlar toplayın ve dikkatli bir şekilde supernatant atın. </li></ol></li><li> -20 ° C de bir gece inkübe sferoplast granül </li></ol> 2. Strep-eklentisi Membran protein saflaştırılması (yem) <ol><li> Buz üzerinde sferoplast pelet yerleştirin. &lt;/ Li&gt; <li> Sferoplast pelet, buz üzerinde çözülme süre boyunca, her bir sferoplast hazırlanması için, 10 ml tampon P3 hazırlar. Yavaşça 10 ml Tris-tampon maddesi içinde 1 mg DNaseI çözülür. Kullanımdan hemen önce 0.1 ml 1 M PMSF ekleyin. </li><li> 6 ml taze hazırlanmış P3 pelet tekrar. Spheroplastlar engellemek amacıyla, her bir patlama ile 1 dakika duraklama ile buz üzerinde sürekli olarak 1 dakika boyunca bir numune dört kez sonikasyon. </li><li> Hasat hücre artıkları için 10 dakika 10,000 x g&#39;de santrifüjleyin örnek. Ultrasantrifüj işlemi, tüp, bir pipet kullanılarak süpernatan aktarın. </li><li> 30 dakika boyunca 100,000 x g&#39;de membran fraksiyonu Pelet. Çözündürülerek olmadan Tris-tampon maddesi içinde dikkatli bir şekilde pelet yıkayın. Bir ince kağıt (örneğin, Kleenex) ile tüp kurutun. Herhangi bir zamanda pelet rahatsız kaçının. </li><li> 1 ml Tris-tampon maddesi içinde dikkatli bir pelet (= zar) yeniden süspanse edin. Örneğin, BCA analizi ile protein konsantrasyonunu belirlemek için, 20 ul bir kısım kullanın. Bu adımda, membranlar şok li dondurulabilirsterlin azot ve -80 ° C&#39;de saklandı </li></ol> 3. Strep-eklentisi Membran yem Protein ve SDS-PAGE ile saflaştırılması <ol><li> Tris-tamponu ile 5 mg / ml membran fraksiyonunun konsantrasyonu protein normalize. Ultrasantrifüj işlemi bir tüp içinde 2.5 ml membran fraksiyonu (5 mg / ml) alın ve zar proteinleri çözündürülmesi amacıyla,% 2&#39;lik bir nihai konsantrasyona ulaşmak için% 20 Triton X-100 ve 0.25 ml ekleyin. Bir mikro-manyetik çubuk ilave edilir ve 1 saat boyunca buz üzerinde karıştırılmıştır. Not: genel olarak, deterjan olarak Triton X-100 kullanılarak iyi sonuçlar vermesine rağmen, bu optimizasyonu için deterjan değiştirmek için yararlı olabilir. Bu bağlamda, ilgili yem zar proteininin fonksiyonel saflaştırılması için kullanılan deterjan ile en iyi sonuçları vardır. </li><li> Adım 3.1 yerine getirirken, 50 ml (Tablo 1) W ve 5 ml tampon E tampon hazırlar. 10 ml 5x tampon W konsantre ml 50 kadar doldurun ve 150 ul% 20 Triton X-100 ekleyin. <ol><li> 1 ml 5x Buffe doldurunr E 10 ml&#39;ye konsantre ve 30 ul% 20 Triton X-100 ilave edin. 8 ml tampon W ile bir 1 ml Strep-taktin superflow yerçekimi akışlı sütuna Denge Not: Bu saflaştırma işlemi sırasında herhangi bir tampon maddesi içinde kritik misel konsantrasyonunun (cmc) üstünde bir konsantrasyonu, 10-kat içinde çözündürme için kullanılan deterjan eklemek için gereklidir. </li></ol></li><li> Çözünmeyen membran fraksiyonu pelet haline getirmek üzere, 30 dakika boyunca 100,000 x g&#39;de çözündürme örnek ve ultrasantrifuju gelen mikro-manyetik çubuk çıkarın. Daha fazla saflaştırma için bir pipet kullanılarak süpernatantı. </li><li> Kolona süpernatant yerleştirin. Sadece yerçekimi akışı ile sütun çalıştırın. Bu yıkama adımı 5x tekrar 5 ml tampon W. ile sütun yıkayın. </li><li> Bu yıkama adımı 4x tekrarlayın 1 ml tampon E. ile membran yem proteinleri Zehir. </li><li> Bir santrifüj filtre birimi ile elüsyon fraksiyonları 2, 3, ve 4-300 ul konsantre edin. </li><li> 50 ul 5x SDS-PAG ile her bir numunenin 200 ul karıştırınE yükleme boyası. 125 ul hacimde her hazırlık ayrıldı. Formaldehit çapraz bağlantıları tersine çevirmek için, 95 ° C&#39;de 20 dakika boyunca her bir preparasyonun bir kısım kaynatın. Numuneler tezgah üstünde en az 10 dakika boyunca oda sıcaklığına kadar soğumasını bekleyin. </li><li> Immunoblotting için uygun olan bir poliakrilamid jel tek bir şerit için yük Her bir numune, 30 ul. Iyi yönlendirme için bir Prestained molekül ağırlığı bir kalem kullanın ve SDS-PAGE 10 çalıştırın. </li></ol> 4. İmmünoblot Analizi Etkileşim Ortaklar Onayla <ol><li> Bir kez, örneğin 3,8 ile yan-kuru bir nitroselüloz zara transfer proteinleri aşama tamamlanır. </li><li> Spesifik etiketleme önlemek için membran engelleyin. % 0.05 Tween-20 (TBS-T) son bir konsantrasyon ile takviye edilmiş Tris-tamponlu tuzlu su içinde hacim başına% 3 ağırlık elde etmek sığır serum albümini (BSA) tartı ile blokaj tamponu hazırlayın. Membran karşılamak için blokaj tamponu yeterli bir hacmi kullanın. Oda sıcaklığında 1 saat için bloke membran. </li><li> Seyreltiknd zamanki gibi av proteini spesifik bir ilk antikor kuluçkaya yatmaktadır. İkinci antikor olarak bir HRP-bağlı antikor kullanarak. </li><li> Yüksek hassasiyet ile bir kemilüminesan algılama kiti kullanılarak immüno geliştirin. Klasik bir film işleme yordamı veya dijital görüntüleme ekipmanı kullanarak sinyalini izleyin. </li></ol> 5. Etkileşim Ortaklar tanımak NanoLC-ESI-MS/MS Yüksek Çözünürlük Deneyleri <ol><li> Belirlenmelidir özel bir antikor, av proteini ya da bilinmeyen bir etkileşim ortakları için kullanılabilir durumda, tanımlama için kütle spektrometresi (MS) kullanılır. Keratin kirlenmesini önlemek amacıyla, protein ayrılması için precasted jelleri kullanın. </li><li> Gümüş, üreticinin protokolüne göre, bir MS-uyumlu bir boyama kiti kullanılarak SDS-PAGE leke. Cam tanklarda tüm boyama ve yıkama adımları gerçekleştirin. </li><li> Tüketim ilgili bantları ve yüksek çözünürlüklü LC / MS 9 bu analiz. </li></ol>

<ol>
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Biz ifşa hiçbir şey yok.

Membran OMURGA analiz onaylamak ve membran proteinlerinin bu noktada bilinmeyen bir etkileşim ortakları tanımlamak için birini sağlayan biyokimyasal bir yaklaşımdır. Membran OMURGA bir Strep-etiketli yem zar proteini saflaştırılması ile formaldehid ile çapraz bağlanması vivo olarak bir araya getirmektedir. Immüno-lekeleme ile kombinasyon tahmin etkileşim ortaklarının onay (Şekil 2) kolaylaştırır. Buna ek olarak, MS analizi ile kombine bilinmeyen etkileşim ortaklarının ...

Bir proteinin işlevini anlamak için onun etkileşim ortaklarını bilmek önemlidir. Çeşitli klasik teknikler çözünür proteinlerin etkileşimi ortaklarının tespiti için kullanılabilir. Ancak, bu teknikler, hidrofobik doğası 4 dolayı zar proteinleri kolaylıkla transfer edilemez. Bu sınırlamayı aşmak için, Membran Strep-protein etkileşim deneyi (Membran-OMURGA) 6 geliştirdik. Bu çözünür proteinler 7 için uygundur olan OMURGA yöntemine dayanmaktadır. Çapraz bağlama maddesi, iki formaldehid avantajlarından zara OMUR faydaları: ilk olarak, formaldehit kolayca nüfuz membranlar ve bu nedenle, bir canlı hücrenin 8&#39;in interactome kesin bir görüntüsünü oluşturur olabilir. İkincisi, formaldehit çapraz bağlantılar 9 kaynatılarak ters olabilir. Burada, bu iki avantajı zar prote sürekli değil, aynı zamanda geçici bir PPI, sadece tanımlamak için kullanılırins 6. Kısaca, Strep-eklentisi, integral zar yem proteinin C-ucuna kaynaştırılır. Membran yem proteini eksprese eden hücreler, çapraz bağlantıları membran proteinine yem proteinleri av formaldehid (Şekil 1) ile birlikte inkübe edilir. Av proteinleri modifikasyonları gerekli değildir. Daha sonra, membran fraksiyonu hazırlanır. Bu nedenle, membran proteinleri proteinleri afinite arıtma kullanarak av proteinleri ile birlikte saf hale deterjan tedavisi ve yem ile eritilir. Daha sonra, çapraz-link kaynatma ile tersine çevrilir ve yem ve eş-elüt av proteinler SDS-PAGE ile ayrılmıştır. Son olarak, av proteinleri bağışıklık beneklenme analizi veya kütle spektroskopisi ile tespit edilebilir.

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			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
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			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
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			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
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			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
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			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
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			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
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membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

Zar proteinleri, hücre canlılığı için gerekli olan ve bu nedenle önemli bir terapötik hedef 1-3 bulunmaktadır. Bu kompleksler 4 işlev için, bunların etkileşimleri belirlemek ve karakterize etmek için yöntemler 5 gereklidir. Bu amaçla, biz Membran-OMURGA 6 denilen Membran Strep-protein etkileşim deney geliştirdi. Bu teknik, Strep-işaretli zar yem proteinin afinite saflaştırma ile geri dönüşümlü çapraz bağlayıcı formaldehit kullanılarak çapraz bağlama, in vivo olarak bir araya getirmektedir. İşlem sırasında, çapraz bağlanmış av proteinleri membran yem protein ile birlikte saf hale getirilir ve daha sonra kaynatılarak ayrılır. Membran omurga kullanılarak zar proteinlerinin protein-protein etkileşimleri (PPI) analiz Bu yüzden, iki ana görevleri icra edilebilir: ilk olarak, immün, tarafından önerilen bir etkileşim ortağının ikinci onay, kütle spektrometrisi analizi ile yeni etkileşim ortaklarının tespiti . Dahası, lafinite ow, geçici PPIs Bu teknik ile tespit edilebilir. Son olarak, Membran-OMURGA membran proteinlerinin PPI tanımlamak için güçlü bir tarama aracı olarak geçerli hale, hemen hemen herhangi bir hücre tipine uyarlanabilir.

Membran OMURGA analiz zar proteinlerinin ortak arıtma ve geçici olarak etkileşimli protein ortakları sağlar. Co-saflaştırma çapraz bağlama ajanı formaldehit kullanılarak elde edilir. Formaldehit konsantrasyonu ve çapraz bağlama süresi: İki parametre spesifik olmayan çapraz bağlantı engellemek için kritik öneme sahiptir. Formaldehid aşırı yeterli ama kullanımı kolay immunoblotting ile kontrol edilebilir. Formaldehit çapraz bağlanmış protein kompleksleri, SDS işlenerek kaynama değil ile ayr?...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

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

Bir yaklaşım, biyokimyasal zar proteinlerinin in vivo protein-protein etkileşimleri (PPI) tanımlamak için tarif edilmektedir. Yöntem, protein çapraz-bağlama, afinite arıtma ve kütle spektrometrisi birleştirir, ve hemen hemen herhangi bir hücre tipi ya da organizma için uyarlanabilir. Bu yaklaşımla, geçici PPI bile tespiti mümkün olur.

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 ...

membrane-spine-biochemical-tool-to-identify-protein-protein

Research

JoVE Journal

Bioengineering

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

13.6K Görüntüleme.  Universität Osnabrück.  Bir yaklaşım, biyokimyasal zar proteinlerinin in vivo protein-protein etkileşimleri (PPI) tanımlamak için tarif edilmektedir. Yöntem, protein çapraz-bağlama, afinite arıtma ve kütle spektrometrisi birleştirir, ve hemen hemen herhangi bir hücre tipi ya da organizma için uyarlanabilir. Bu yaklaşımla, geçici PPI bile tespiti mümkün olur. 

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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Nükleik Asitler, Proteinler ve Küçük Moleküller Reagentless Algılama Elektrokimyasal DNA Biyosensörler Fabrikasyon

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Biyomühendislik

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Negatif Leke EM, Proteinler ve makromoleküler kompleksler görselleştirme: Izgara Hazırlık Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Plasenta karşısında Ksenobiyotik ve Nanomalzemelerin Ulaştırma Hızı belirlenmesi kullanarak<em&gt; Ex vivo</em&gt; İnsan Plasental Perfüzyon Modeli

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Protein A Membran Adsorberleri ve Kolaylaştırılmış bir İş Akışı Kullanarak Kimerik İnsan Fc Ekspresyon Proteinlerinin İzolasyonu ve Karakterizasyonu

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lüminesans Rezonans Enerji memeli hücrelerinde ifade Membran Proteinleri Konformasyonel Değişiklikleri Eğitim transfer

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Aracı Olarak bir lipozomal-kapsüllü Yakın-Kızılötesi floroforun Floresan-söndürme<em&gt; İn Vivo</em&gt; Optik Görüntüleme

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Sandviç gibi mikroçevrelerde Hücre / Malzeme Etkileşimleri Harness

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D Etkileşimleri Çalışmaları 3D Kollajen jeller ve Mikrokanallarda Hazırlanması<em&gt; İn Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA nanotüpler Semiflexible polimerler eğitim için çok yönlü bir araç olarak

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...