Bu çalışma Sağlık Araştırma Kanada Enstitüsü tarafından desteklenmiştir. CSC Doğa Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Konseyi doktora sonrası burs tarafından finanse edildi. FD bir Tier II Kanada Araştırma Başkanı olduğunu.

<html> Genel Sulandırma Süreci Sulandırma işlemi deterjan çözülmüş fosfolipidlerin mevcudiyetinde saflaştırılmış MalFGK 2 kompleks ile membran protein iskele (MSP) ve karıştırma başlatılır. Adım Bio-boncuk veya Amberlite (Şekil 1) olarak adlandırılan bir emici malzeme ile polistiren deterjan yavaş kaldırılması tarafından takip edilir. Otomatik montaj işlemi çünkü hidrofobik fosfolipidler, MalFGK 2 karmaşık ve MSP amfipatik proteinin yüzeyi ile apolar etkileşim büyük olasılıkla oluşur. Nihai ürün MalFGK 2 kompleks MSP sarma iki molekül yapılmış bir disk biçiminde bir parçacıktır. Partiküller ultra-santrifüj ile analitik boyut-dışlama kromatografisi ile adducts ve agregadan ayrılır. Partiküller doğal-jel elektroforezi ve dinamik ışık saçılması spektroskopisi ile karakterize edilmiştir. Membran başlığı &quot;&gt; 1. Hazırlama İskele Protein, MSP <ol><li> His-etiketli MSP (versiyon MSP1D1 1) E. plazmid pMSP1D1 üretilir coli, 37, 3 saat boyunca 0.5 mM izopropil β-D-1-thiogalactopyranoside ile OD 600 ~ 0.5 indüklenen BL21 (DE3) ° C. </li><li> Hücreler 10 dakika 4 ° C&#39;de 5.000 x g santrifüj ile toplanır ve 100 uM fenilmetansülfonil flor içeren TSG10 tampon içinde tekrar süspanse edilir. </li><li> Hücreler 8,000 psi ile bir Fransız pres (3x) ile lize edilir ve erimez madde 4 ° C sıcaklıkta 10 dakika için 5000 x g&#39;de santrifüj ile çıkarılır MSP içeren çözünür kesir 45 dakika süreyle 125,000 xg ultra santrifüj yoluyla izole edilir. </li><li> MSP nikel-şelat kromatografi TSG10 tampon içinde ~ 1.5 ml Ni Sepharose HP kullanılarak saflaştırılır. Kirletici maddelerin, 50 mM imidazol içeren tampon TSG10 ile yıkandıktan. MSP 600 mM TSG10 tampon ile elüt edilmiştirimidazol. Saflaştırılmış protein TSG10 tampon içinde diyaliz edildi ve ~ 10-15 mg / ml &#39;lik bir protein konsantrasyonu -70 ° C de saklanır. </li></ol> 2. MalFGK 2 Kompleksi hazırlanması <ol><li> MalFGK 2 kompleksinin Malk, C-terminalinde His-etiketli E. plazmid pBAD22-FGK eksprese edilir coli BL21 (DE3) ve 37 de, 3 saat boyunca% 0.2 L-arabinoz ile OD 600 ~ 0.5 indüklenen ° C. </li><li> Adım 1.3 &#39;de olduğu gibi hücrelerinin lisisi ve santrifüj sonrasında, zar pelet 5 mg / ml&#39; lik bir nihai konsantrasyonda TSG20 tampon içinde tekrar süspanse edilir. Malzeme hafif sallama ile 4, 3 saat boyunca% 1 w / v n-dodesil-β-maltopyranoside (DDİ) ° C ile çözündürülmüş edilir. </li><li> Çözünmeyen malzeme adım 1.3 &#39;de olduğu gibi ultra-santrifüj ile çıkarıldı ve yüzey maddesi içeren bir çözünür MalFGK 2 kompleksinin toplanan ve adım 1,4 olarak saflaştırılmış, ama diyalize olmadan edilir. </li><li> Further arıtma 0.5 ml / dak &#39;lık bir akış hızında, bir Superdex 200 HR 10/300 kolon üzerinde TSGD tampon içinde, jel filtrasyon kromatografisi ile elde edilir. </li></ol> 3. Fosfolipidler hazırlanması <ol><li> Bir E. coli, toplam lipid kloroform içinde çözüldü özü vidalı kapaklı mikrofüj tüpler içinde 1.000 nmol alikotları ayrılır. Çözücü, bir azot akımı altında buharlaştırıldı yumuşak ve bir vakumlu desikatör içinde daha ileri gece boyunca kurutulur. </li><li> Lipid filmi 5 nM son konsantrasyonda TS tampon içinde çözülmüş ve kuvvetlice vortekslenmiş ve sonike edilmiştir. Çözünmüş lipidler nedeniyle sulu TS tampon genel çözülmezlik hafifçe opak görünecektir, ancak askı kalmalıdır. DDİ çözelti berrak hale geldiği% 0.5 (~ 10 mm), ve bir nihai konsantrasyon ilave edilir. </li><li> Lipit karışımı bir su banyosuna yerleştirilir ve nabız ~ 5 sn için 5 kez sonike edilir. Lipid karışımı 1 çiş bir süre için 4 ° C sıcaklıkta saklanırk. </li></ol> 4. Bio-Boncuk hazırlanması <ol><li> Yaklaşık 10-15 ml (kuru cilt) Bio-Boncuk 50 ml&#39;lik bir tüp yerleştirilir. </li><li> Boncuklar art arda% 100 metanol,% 95 etanol, milliQ H2O, ve son olarak da TS tamponu (iki defa her biri), 50 ml ile yıkanır. </li><li> Yıkanmış boncuklar 4 saklanır ° C ~ 10 ml tampon içinde TS. </li></ol> 5. Nanodisc Sulandırma <ol><li> MSP ~ 7 mg / ml (~ 0.3 mM) nihai konsantrasyonu TSGD tamponu içinde seyreltilir. </li><li> MSP: TSGD tampon içinde 1:3:60 veya 1:3:400 lipid oranının saflaştırılmış MalFGK 2 kompleks ~ 2 nmol bir protein karıştırılmıştır. Nihai konsantrasyon 6 uM MalFGK 2, 18 uM, 360 uM MSP lipidler (1:3:60) ya da 2.4 mM lipidler (1:3:400) &#39;dir. Son hacim 300 ul olup. Nihai DDM konsantrasyonu% 0.08 (~ 1.6 mM) &#39;dir. Gliserol nihai konsantrasyon lipit miktarına bağlı olarak değişir, ancak ilave etrafında% 5-10 v / v kalır </li&gt; <li> ~ Bio-boncuk süspansiyonu 50 ul tüpüne ilave edilir ve karışım, 4 ° C&#39;de bir sallanan bir masa üzerinde bir gece inkübe edilir </li><li> Boncuklar yerçekimi ile sedimente edilir ve çözelti, mümkün olduğu kadar çok Biyo-Boncuk önlemek için dar bir ucu ile pipetle konulur. </li><li> Çökelek Büyük 20 dakika için 100,000 x g ultra-santrifüj ile çıkarılır. Diskler TSG10 tampon içinde adım 2.4 &#39;de olduğu gibi, jel filtrasyon kromatografisi ile saflaştırılır. Diskleri ihtiva eden fraksiyonlar bir araya getirilmiştir ve bir kısım hazırlanması kararlılık test etmek için aynı jel süzme sütunu üzerine, yeniden enjekte edilmelidir. </li><li> Saflaştırılmış disklerin uzun bir süre -70 ° C&#39;de saklanabilir. Buz üzerinde eritme sonra, diskler, potansiyel çökeltileri uzaklaştırmak için adım 5.5 gibi ultra-santrifüj tabi tutulmalıdır. Bir kısım önemli agregasyonu veya yağış çözdürme sırasında meydana vermedi sağlamak için jel filtrasyon kromatografisi ile analiz edilmelidirsüreç. </li></ol> 6. Yerli Jel Elektroforezi <ol><li> Nanodiscs 1 uM (~ 0.2 mg / ml) ile sulandırma (Şekil 3A) kalitesini ölçmek için doğal PAGE 6, 7 (Tris-HCl, pH 8.8,% 4-12) ile analiz edilir. </li><li> 2-kompleks MalFGK için erkek bağlayıcı (1 uM) nanodiscs yılında yeniden yapılandırılmış, ayrıca yerel-PAGE (Şekil 3A) tarafından değerlendirilir. </li><li> Elektroforez sonra, jel 10 dakika için Coomassie mavisi ile boyandı, ve ~ 1 saat boyunca destained. </li></ol> 7. Dinamik ışık saçılımı (DLS) <ol><li> Nanodiscs 0.1 ml / dak &#39;lık bir akış hızında, bir Superdex 200 HR 10/300 kolonu kullanılarak TSG10 tampon içinde adım 2.4&#39; de olduğu gibi, jel filtrasyon kromatografisi ile saflaştırılır. Nanodiscs ihtiva eden fraksiyonlar toplanmış ve bir Amicon santrifüj filtre ile yaklaşık 10 mg / ml &#39;ye konsantre edilmiştir. </li><li> Örnek kullanarak DLS ile analizden önce (0.22 mikron filtre) iki kez filtre edilirBir 1 ul iç hacmi kuvars küvetine bir Dynapro nanostar enstrüman (Wyatt Technology). Veriler, çap ve parçacıkların moleküler ağırlığı tahmin etmek DİNAMİK yazılım (Wyatt Technology) ile monte edilir. </li></ol> 8. ATPaz Ölçümleri <ol><li> MalFGK 2-Sulandırılan nanodiscs arasında ATPaz aktivitesi kolorimetrik 8 kullanılarak belirlenir. </li><li> Saflaştırıldı diskleri ve 1 mM ATP 1 uM, 20 dakika boyunca erkek ve 37 ° C de inkübe artan miktarları ile karıştırılır. Inorganik fosfat sürümü 660 nm&#39;de ölçülür. </li><li> Serbest fosfat miktarı, bir fosfor Standart çözelti ile oluşturulan standart bir eğri ile karşılaştırılmıştır. </li></ol> 9. Temsilcisi Sonuçlar Nanodiscs jel filtrasyon kromatografisi (Şekil 2A, sol) ile arıtılır. Kromatogramı göstermektedir ki sulandırılmış d çoğunluğuISCS (siyah iz) tek bir zirve olarak elute, oysa aşırı lipid (kırmızı iz) boşluk hacminde elute olan ve geniş zirveleri bir dizi olarak yapılmış diskler. Nanodiscs kalitesi daha fazla doğal jel elektroforezi ve dinamik ışık saçılması spektroskopisi (DLS) ile analiz edilir. Lipidlerin aşırı varlığında Sulandırılan bu bir smear (Şekil 2A, sağ) olarak göç ise Düzgün Sulandırılan diskler jel üzerinde keskin bir bant olarak göç ederler. DLS disk ile analiz nüfus 11.4 nm (Şekil 2B), bir ortalama çapa sahip homojen olduğunu göstermektedir. Sulandırılan DLS disk yaklaşım esas 215 kDa bir görünür moleküler ağırlığa sahiptir. Aşırı lipid varlığında sulandırılmış Örnekler yaygın olarak homojen olmayan örnekler için tipik olan, 100 nm yarı çapları etrafında dağıtılmış ekranı. MalFGK 2 kompleksinin kalitesi ATPaz tarafından yerli jel elektroforezi ve faaliyet değerlendirilirölçümleri (Şekil 3). Maltoz bağlayıcı protein Erkek MalFGK 2 taşıyıcı 9, 10 yüksek afinite ile bağlanır. Kullanılması olmayan denatüre edici jel elektroforezi, erkek ve MalFGK 2 (Şekil 3A) arasında bir kompleks tespit etmek mümkündür. Erkek Malk 2 ATPaz aktivitesinin uyarılması Şekil 3B &#39;de gösterilmiştir. <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Şekil 1"/> Şekil 1. Sulandırma protokolü için tipik akış şeması. <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Şekil 2,"/> Şekil 2. Nanodisc hazırlanması. A. Kalite Kontrol Veya yüksek yağ oranı (1/3/400; kırmızı iz); nanodiscs Jel filtrasyon analizi (Superdex 200 HR 10/300 sütun) düşük yağ oranı (siyah iz 1/3/60) de sulandırılır. Aynı diskin hazırlık Native jel elektroforezi. MolkDa yılında ecular ağırlığı işaretleri gösterilir. Aynı diskin hazırlanması B. Dinamik ışık saçılımı analiz. <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Şekil 3"/> Şekil 3,. MalFGK 2-nanodisc parçacıklar analizi. C. Male jel kayma analizi MalFGK 2-nanodisc parçacıkların artan miktarlarda ile inkübe edildi. MalFGK B. ATPaz aktivitesi 2-nanodisc Erkek konsantrasyonunun bir fonksiyonu olarak parçacıklar.

<ol>
	<li>Denisov, I. G., Ginkova, Y. V., Lazarides, A. A., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+monodisperse+phospholipid+bilayer+Nanodiscs+with+controlled+size.">Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.</a> J. Am. Chem. Soc. 126, 3477-3487 (2004).</li><li>Boldog, T., Grimme, S., Li, M., Sligar, S. G., Hazelbauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+separate+chemoreceptor+oligomeric+states+and+reveal+their+signaling+properties.">Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.</a> Proc. Natl. Acad. Sci. USA. 103, 11509-11514 (2006).</li><li>Bass, B. J., Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homotropic+cooperativity+of+monomeric+cytochrome+P450+3A4+in+a+nanoscale+native+bilayer+environment.">Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.</a> J. Biol. Chem. 282, 7066-7076 (2007).</li><li>Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+unravel+the+interaction+between+the+SecYEG+channel+and+its+cytosolic+partner+SecA.">Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.</a> EMBO J. 26, 1995-2004 (2007).</li><li>Mi, L. -. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+stability+of+the+epidermal+growth+factor+receptor+in+detergent+micelles+and+phospholipid+nanodiscs.">Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.</a> Biochemistry. 47, 10314-10323 (2008).</li><li>Sch&#228;gger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217, 220-230 (1994).</li><li>Dalal, K., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+SecY+translocon+in+Nanodiscs.">Reconstitution of the SecY translocon in Nanodiscs.</a> Methods Mol. Biol. 619, 145-156 (2010).</li><li>Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., Candia, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+assay+for+nanomole+amounts+of+inorganic+phosphate.">An improved assay for nanomole amounts of inorganic phosphate.</a> Anal. Biochem. 100, 95-97 (1979).</li><li>Davidson, A. L., Dassa, E., Orelle, C., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+evolution+of+bacterial+ATP-binding+cassette+systems.">Structure, function and evolution of bacterial ATP-binding cassette systems.</a> Microbiol. Mol. Biol. Rev. 72, 317-364 (2008).</li><li>Bordignon, E., Grote, M., Schneider, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maltose+ATP-binding+cassette+transporter+in+the+21st+century-towards+a+structural+dynamic+perspective+on+its+mode+of+action.">The maltose ATP-binding cassette transporter in the 21st century-towards a structural dynamic perspective on its mode of action.</a> Mol. Microbiol. 77, 1354-1366 (2010).</li><li>Alvarez, F. J., Orelle, C., Davidson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+an+ABC+transporter+for+use+in+electron+paramagnetic+resonance+spectroscopy.">Functional reconstitution of an ABC transporter for use in electron paramagnetic resonance spectroscopy.</a> J. Am. Chem. Soc. 132, 9513-9515 (2010).</li><li>Ritchie, T. K., Grinkova, Y. V., Bayburt, T. H., Denisov, I. G., Zolnerciks, J. K., Atkins, W. M., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+in+phospholipid+bilayer+Nanodiscs.">Reconstitution of membrane proteins in phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 464, 211-231 (2009).</li><li>Gl&#252;ck, J. M., Koenig, B. W., Willbold, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+allow+the+use+of+integral+membrane+proteins+as+analytes+in+surface+plasmon+resonance+studies.">Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.</a> Anal. Biochem. 408, 46-52 (2011).</li><li>Wan, C. -. P. L., Chiu, M. H., Wu, X., Lee, S. K., Prenner, E. J., Weers, P. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein-induced+conversion+of+phosphatidylcholine+bilayer+vesicles+into+nanodisks.">Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks.</a> Biochim. Biophys. Acta (BBA). 1808, 606-613 (2011).</li><li>Nath, A., Trexler, A. J., Koo, P. K., Miranker, A. D., Atkins, W. M., Rhoades, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+spectroscopy+using+phospholipid+bilayer+Nanodiscs.">Single-molecule fluorescence spectroscopy using phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 472, 89-117 (2010).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+P450+in+Nanodiscs.">Cytochromes P450 in Nanodiscs.</a> Biochim. Biophys. Acta. 1814, 223-229 (2011).</li><li>Zhang, X. X., Chan, C. S., Bao, H., Fang, Y., Foster, L. J., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+and+SILAC-based+mass+spectrometry+to+identify+a+membrane+protein+interactome.">Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome.</a> J. Proteome Res. , (2011).</li></ol>

Çıkar çatışması ilan etti.

Biz nanodiscs içine maltoz taşıyıcı sulandırılması için basit bir prosedür açıklanmaktadır. Taşıyıcı ATPaz aktif ve çözünen bağlayıcı ortağı erkek ile etkileşim (Şekil 3) yeniden olabilir. Nanodiscs içine taşıyıcı başarılı rekonstitüsyon ek biyofiziksel ve biyokimyasal analiz için yolu açın. Özellikle ilgi Of Malk ATPaz ve deterjan maltoz taşımacılık faaliyeti, lipozom ve nanodiscs sistematik analiz olacaktır. ABC taşıyıcıları taşıma döngüsü sıras?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Amicon Ultra-4 50K santrifüj filtre </td><td> Millipore </td><td> UFC805008 </td><td> Uygun kullanımı için üreticinin protokol izleyin </td></tr><tr><td> Bio-Boncuk SM-2 Adsorban </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli total lipid </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Kloroform içinde çözülmüş, bir organik çözücü uygun olarak işlemek </td></tr><tr><td> Ni Sepharose HP reçine </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Fosforlu standart çözelti </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE Healthcare </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tablo I. Belirli reaktifler. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Isim </td><td> Kompozisyon </td><td> Yorumlar </td></tr><tr><td> DDM stok </td><td> % 10 w / v DDM </td><td> -20 ° C&#39;de milliQ su ve mağaza süspanse </td></tr><tr><td> MalFGK 2 stok </td><td> 1-2 mg / ml &#39; 50 mM Tris-HCl, pH7.9 100 mM NaCl % 10 h / h gliserol % 0.03 w / v DDM </td><td> -70 ° C&#39;de saklayın sonra purification </td></tr><tr><td> MSP stok </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl % 10 h / h gliserol </td><td> -70 ° C&#39;de saklayın &lt;1 ml alikotları arıtma sonrası ve aşırı donma / çözülme döngüleri önlemek </td></tr><tr><td> Fosfolipid stok </td><td> 5 nM E. coli lipidler toplam % 0.5 w / v (10 mM) DDM 50 mM Tris-HCI, pH 7.9 50 mM NaCl </td><td> 1 hafta boyunca 4 ° C&#39;de depolayın </td></tr><tr><td> TS tampon </td><td> 50 mM Tris-HCI, pH 7.9 50 mM NaCl </td><td> 4 ° C&#39;de saklayın </td></tr><tr><td> TSG10 tampon </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl % 10 h / h gliserol </td><td> 4 ° C&#39;de saklayın </td></tr><tr><td> TSG20 tampon </td><td> 50 mM Tris-HCl, PH8 100 mM NaCl % 20 h / h gliserol </td><td> 4 ° C&#39;de saklayın </td></tr><tr><td> TSGD tampon </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl % 10 h / h gliserol % 0.03 w / v DDM </td><td> 4 ° C&#39;de saklayın ve sadece kullanmadan önce DDM ekleyin </td></tr><tr><td colspan="3"> Tablo II. Çözüm tarifleri. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

Nanodisc bir diskoidal parçacık (~ 10-12 nm büyük) o tuzağa membran proteinlerinin fosfolipid çift tabakası küçük bir yama içine. Olduğunu Nanodisc özellikle ligand-reseptör etkileşimlerinin bağlamında, zar proteinleri çalışmak için özellikle çekici bir seçenek. Yöntem Sligar tarafından ve arkadaşları, apolipoprotein A1 türetilen bir tasarlanmış çok a-sarmal iskele protein amfipatik özelliklerine dayanmaktadır. Kutup bölgeleri sulu ortamda karşı karşıya iken iskele proteinin hidrofobik yüzleri iki tabakalı lipid ve yağ açil yan zincirleri ile etkileşim. Partikülleri, küçük homojen değildir ve suda çözünebilir olduğu için nanodiscs içinde zar proteinlerinin analizi lipozom göre önemli avantajlara sahiptir. Buna ek olarak, normal olarak çözünür proteinler için ayrılmış biyokimyasal ve biyofiziksel uygulanan yöntemler, ve zarın her iki tarafında olabilir. Bu görsel protokol, biz iyi bir karakter bir adım-adım sulandırma sunmakkarekterize bakteriyel ABC taşıyıcı, erkek-MalFGK 2 kompleksi. Diskin oluşum deterjan ilerleyen çıkarılması sırasında meydana gelen hidrofobik etkileşimler bağlı olarak bir öz montaj işlemdir. Biz gerekli adımları açıklamak ve agrega ve büyük polidispers lipozom benzeri partiküllerin oluşumunu sınırlamak için doğru protein-to-lipid oranı seçmenin önemini vurgulamak. Basit kalitesi tür diskler düzgün sulandırılmış oylandı emin jel filtrasyon kromatografisi, yerli jel elektroforezi ve dinamik ışık saçılma spektroskopisi olarak denetler.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscs fosfolipid çift tabakası küçük bir yama içine membran proteinleri dahil küçük diskoid parçacıklardır. Biz bir disk içine MalFGK2 taşıyıcı adım adım kuruluş gösteren görsel bir protokol sağlar.

Nanodisc Lipid Parçacıklar içine bir ABC Transporter Sulandırma için bir adım-adım yöntemi

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

a-step-step-method-for-reconstitution-an-abc-transporter-into

Research

JoVE Journal

Biology

25.3K Görüntüleme.  University of British Columbia. Nanodiscs fosfolipid çift tabakası küçük bir yama içine membran proteinleri dahil küçük diskoid parçacıklardır. Biz bir disk içine MalFGK2 taşıyıcı adım adım kuruluş gösteren görsel bir protokol sağlar.

Video: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

İfade, Deterjan çözünürleştirme ve Membran Transporter, MexB ilaca Direnç Protein Saflaştırılması

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Biyoloji

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Active T. in vitro Sulandırma castaneum Telomeraz

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Görüntüleme için Basit Bir Yöntem Arabidopsis Infiltratif Görüntüleme Orta Perfluorodecalin kullanarak bırakır.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Yapısal ve İşlevsel Araştırmalar için Lipid Membranlar bir Kv Kanal Sulandırma

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Bir transmembran proteinin yeniden oluşturulması, Mikroskop ve yama kelepçe Çalışmaları Dev tek-katmanlı keseler içine Voltaj kapılı iyon kanalı, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Prosedür bitki özleri gelen Dağınık Parçacıkların çıkarması için Flokülantlar Verimliliği değerlendirin

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

Transcriptionally düzenlenmiş uyku güzellik sistem entegrasyon arızalı Lentiviral vektör paketlenmiş bir verimli Vitro transpozisyonu yöntemle

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Bir doku Yöntem yağ dokusu üç boyutlu görüntüleme için temizlenmesi

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

Toksisite Testi için Havadaki Parçacıklara Gerçekçi, Tekrarlanan Soluma Maruziyeti için Hava Sıvısı Arayüzü Bronşiyal Epitel Modeli

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...