We thank Barbara Windschiegl for her help in establishing SOPs; Dennis Remme for his work on the NanoCalcFX software and Alina Kollmannsperger, Markus Braner and Milan Gerovac for helpful suggestions on the manuscript. The German-Israeli Project Cooperation (DIP) provided by the DFG and the Federal Ministry of Education and Research to R.T., as well as the Federal Ministry of Economics and Technology (ZIM R&amp;D Project) to R.T. and Nanospot GmbH supported this work.

<ol>
	<li>Yildirim, M. A., Goh, K. I., Cusick, M. E., Barabasi, A. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-target+network.">Drug-target network.</a> Nat Biotechnol. 25, 1119 (2007).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Osaki, T., Suzuki, H., Le Pioufle, B., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+simultaneous+measurements+of+single-molecule+translocation+in+&#945;-hemolysin+nanopore+array.">Multichannel simultaneous measurements of single-molecule translocation in &#945;-hemolysin nanopore array.</a> Anal. Chem. 81, 9866-9870 (2009).</li><li>Carrillo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+membrane+capacitance+measurements+for+studying+endocytosis+and+exocytosis+in+yeast.">High-resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast.</a> Traffic. , (2015).</li><li>Giacomini, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+transporters+in+drug+development.">Membrane transporters in drug development.</a> Nat. Rev. Drug Discov. 9, 215-236 (2010).</li><li>Castell, O. K., Berridge, J., Wallace, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+membrane+protein+inhibition+by+optical+ion+flux+in+a+droplet+interface+bilayer+array.">Quantification of membrane protein inhibition by optical ion flux in a droplet interface bilayer array.</a> Angew. Chem. Int. Ed. 51, 3134-3138 (2012).</li><li>Zollmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+liposome+analysis+of+peptide+translocation+by+the+ABC+transporter+TAPL.">Single liposome analysis of peptide translocation by the ABC transporter TAPL.</a> Proc. Natl. Acad. Sci. U.S.A. 112, 2046-2051 (2015).</li><li>Tamm, L. K., McConnell, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+phospholipid+bilayers.">Supported phospholipid bilayers.</a> Biophys. J. 47, 105-113 (1985).</li><li>Sackmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+membranes:+scientific+and+practical+applications.">Supported membranes: scientific and practical applications.</a> Science. 271, 43-48 (1996).</li><li>Castellana, E. T., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+supported+lipid+bilayers:+From+biophysical+studies+to+sensor+design.">Solid supported lipid bilayers: From biophysical studies to sensor design.</a> Surf. Sci. Rep. 61, 429-444 (2006).</li><li>Wagner, M. L., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethered+polymer-supported+planar+lipid+bilayers+for+reconstitution+of+integral+membrane+proteins:+silane-polyethyleneglycol-lipid+as+a+cushion+and+covalent+linker.">Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker.</a> Biophys. J. 79, 1400-1414 (2000).</li><li>Naumann, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+polymer-supported+phospholipid+bilayer:+tethering+as+a+new+approach+to+substrate-membrane+stabilization.">The polymer-supported phospholipid bilayer: tethering as a new approach to substrate-membrane stabilization.</a> Biomacromolecules. 3, 27-35 (2002).</li><li>Weiskopf, D., Schmitt, E. K., Kl&#252;hr, M. H., Dertinger, S. K., Steinem, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-BLMs+on+highly+ordered+porous+silicon+substrates:+Rupture+process+and+lateral+mobility.">Micro-BLMs on highly ordered porous silicon substrates: Rupture process and lateral mobility.</a> Langmuir. 23, 9134-9139 (2007).</li><li>Watanabe, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrayed+lipid+bilayer+chambers+allow+single-molecule+analysis+of+membrane+transporter+activity.">Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity.</a> Nat. Commun. 5, (2014).</li><li>Stamou, D., Duschl, C., Delamarche, E., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembled+Microarrays+of+Attoliter+Molecular+Vessels.">Self-Assembled Microarrays of Attoliter Molecular Vessels.</a> Angew. Chem. Int. Ed. 115, 5738-5741 (2003).</li><li>Lohr, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Liposomes+Used+to+Study+the+Activity+of+Individual+Transporters.">Single Liposomes Used to Study the Activity of Individual Transporters.</a> Biophysical Journal. 106, 229a (2014).</li><li>Dunlop, J., Bowlby, M., Peri, R., Vasilyev, D., Arias, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+electrophysiology:+an+emerging+paradigm+for+ion-channel+screening+and+physiology.">High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology.</a> Nat. Rev. Drug Discov. 7, 358-368 (2008).</li><li>Milligan, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robotic+multiwell+planar+patch-clamp+for+native+and+primary+mammalian+cells.">Robotic multiwell planar patch-clamp for native and primary mammalian cells.</a> Nat. Protoc. 4, 244-255 (2009).</li><li>Soga, N., Watanabe, R., Noji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attolitre-sized+lipid+bilayer+chamber+array+for+rapid+detection+of+single+transporters.">Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters.</a> Sci. Rep. 5, (2015).</li><li>Kleefen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+parallel+single+transport+recordings+on+nanopore+arrays.">Multiplexed parallel single transport recordings on nanopore arrays.</a> Nano Lett. 10, 5080-5087 (2010).</li><li>Wei, R., Gatterdam, V., Wieneke, R., Tamp&#233;, R., Rant, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+sensing+of+proteins+with+receptor-modified+solid-state+nanopores.">Stochastic sensing of proteins with receptor-modified solid-state nanopores.</a> Nat. Nanotechnol. 7, 257-263 (2012).</li><li>Urban, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+parallel+transport+recordings+on+a+membrane-on-nanopore+chip+at+single+molecule+resolution.">Highly parallel transport recordings on a membrane-on-nanopore chip at single molecule resolution.</a> Nano Lett. 14, 1674-1680 (2014).</li><li>Kusters, I., Van Oijen, A. M., Driessen, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-on-a-Chip:+Microstructured+Silicon/Silicon-Dioxide+Chips+for+High-Throughput+Screening+of+Membrane+Transport+and+Viral+Membrane+Fusion.">Membrane-on-a-Chip: Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion.</a> ACS Nano. 8, 3380-3392 (2014).</li><li>Hansen, J. S., Thompson, J. R., He&#769;lix-Nielsen, C., Malmstadt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+directed+intrinsic+membrane+protein+segregation.">Lipid directed intrinsic membrane protein segregation.</a> J. Am. Chem. Soc. 135, 17294-17297 (2013).</li><li>Heinemann, F., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Micrometer-Sized+Free-Standing+Membranes.">Preparation of Micrometer-Sized Free-Standing Membranes.</a> ChemPhysChem. 12, 2568-2571 (2011).</li><li>Lazzara, T. D., Carnarius, C., Kocun, M., Janshoff, A., Steinem, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+attoliter-sized+compartments+using+fluid+pore-spanning+lipid+bilayers.">Separating attoliter-sized compartments using fluid pore-spanning lipid bilayers.</a> ACS nano. 5, 6935-6944 (2011).</li><li>Winterhalter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Black+lipid+membranes.">Black lipid membranes.</a> Curr. Opin. Colloid Interface Sci. 5, 250-255 (2000).</li><li>Ko&#231;er, A., Walko, M., Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+utilization+of+reversible+and+irreversible+light-activated+nanovalves+derived+from+the+channel+protein+MscL.">Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL.</a> Nat. Protoc. 2, 1426-1437 (2007).</li></ol>

The authors declare no competing financial interests.

ここで紹介する手法は、膜タンパク質輸送の高度な並列分析を可能にします。再構成された膜タンパク質系は、直接、理論的にすべての膜輸送体またはチャネル可能の適応を行う、バイオチップに適用することができます。輸送解析は、唯一の直接蛍光変化（蛍光体の転座または蛍光標識された基質）または間接的な蛍光変化（pH感受性色素、二次酵素反応）のいずれかを介して、蛍光読み?...

膜タンパク質の分析は、過去20年間で基礎および医薬品の研究のための関心が高まっになっています。新規薬剤の開発は、現在、制限要因の一つである、新たな標的の同定および詳細な特性に依存します。すべての薬物標的の約60％が膜タンパク質1であるという事実は、最も重要なその機能を解明するための技術の開発を行います。 4 -過去には、起電チャンネルおよびトランスポーターの研究のための技術は、多数の2で開発されてきました。逆における非起電性基板は、より多くの困難な課題を提示します。彼らはカスケード5のシグナル伝達に重要な受容体などの細胞膜と機能を横切る溶質および栄養素のフラックスを制御するように、彼らは、しかしプライム薬物標的としての特別な関心のです。 かなりの努力がトンの開発に置かれていますechniques膜輸送タンパク質6、7の機能を研究します。いくつか例を挙げると固体担持脂質二重層、繋留二重層11、12、microblackの脂質膜13、14およびネイティブベシクルアレイ15、16を含む10、 -固体支持膜を用いたシステムでは、このフィールド8として最も有望なツールを浮上しています。それらのいくつかは、市販のセットアップ17、18のようにしてもご利用いただけます。いくつかの実施例は、高度に並列に14、19、スクリーニング用途のための前提条件で単一の膜タンパク質を研究する能力を組み合わせること公開されています。しかし、これらの方法はほとんどの産業環境への基礎研究から橋渡しません。困難は、多くの場合、自動化するシステムの能力は、コストのかかる生産および/または面倒な準備にあります。アプローチoを上記の全ての障害をvercomingすることは、最終的な目標です。 22 -ここで紹介する手法は、単一のタンパク質レベル20に制御された環境で、in vitroで膜チャネルおよびトランスポーターを研究するために開発されました。 26または黒色脂質膜27 - LUVをに精製された膜タンパク質の再構成は、はるかにGUVs 23で同等のアプローチよりも確立されています。二重層の形成は自己集合プロセスを介して行われている場合、それらは直接チップ表面に適用することができます。ナノ多孔性チップ（ 図 1）のガラスボトム設計により、システムの簡単な自動化を可能にする空気顕微鏡を可能にします。電動ステージとの組み合わせで複数のチップを、分析のために密封された空洞の数千人を含むビューの各フィールドで、同時に測定することができます。 図 1。多重化されたナノ細孔のバイオチップの設計 A）は、シリコン・オン・インシュレータ（SOI）ウエハを反応性イオンエッチングにより構成されています。約1150個々のチップは、同一の特性と品質。B）各チップは、ナノ開口を備え25万個々のマイクロキャビティを備えるとともに、各ウェハから製造されます。スケールバー：200μmのC）各キャビティは、マルチスペクトル蛍光読み出しを経由してアドレス可能です。 intransparent上層倒立蛍光顕微鏡とバイオチップの互換性行うブロックバッファリザーバからの蛍光シグナルを、D）原子間力顕微鏡（AFM）イメージングが均一に配置された孔の開口部および3.6ナノメートルの二酸化ケイ素層の表面粗さを（明らかに、N = 40）小胞の融合のために最適。スケールバー：5μmのE）走査型電子マイクroscopy（SEM）画像は、シリコンチップ内のフェムトリットルの空洞へのアクセスを可能にするナノ細孔の断面を示しています。この数字は21から許可を得て再利用した。 <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> すべてのデータ分析は、エンドユーザのための無制限のアクセスを保証するためにフリーウェアを使用して実行されます。時系列は、フリーの画像処理ソフトウェアとバッチ処理と曲線の複数の蛍光チャネルおよび何千も大きなデータセットの単純な相関関係を可能にするカスタムビルド曲線分析ソフトウェアを用いて分析します。 このプロトコルで使用されるモデルタンパク質は、E由来大コンダクタンス（MSCL）チャネルタンパク質の機械受容チャネルであります大腸菌 。それは、本質的に浸透圧ショックを放出する弁として機能するが、合理的にSYNT設計ように変更されましたhetic機能は、共有チャネルの収縮側に取り付けることができます。共有結合した活性化剤（MTSET）の電荷反発を経由してチャンネルがナノバルブを作成、開くようにトリガされます。イオン、水、小さなタンパク質だけでなく、小型の蛍光団のような小分子は、チャネルを介して透過することができます。ここでは、タンパク質は、タンパク質媒介転座を検出するシステムの能力を実証するためのモデルとして使用されます。

<table border="0" cellpadding="0" cellspacing="0" width="1093">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Reagent</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">[2-(Trimethylammonium)ethyl] 
			methanethiosulfonate</td>
			<td style="width:232px;">Toronto Research Chemicals Inc.</td>
			<td style="width:305px;">T792900</td>
			<td>MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 &#176;C. Use immediately (30 minutes) after solubilization in buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">1 ml gas-tight syringe</td>
			<td style="width:232px;">Hamilton</td>
			<td style="width:305px;">#1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">10 ml round flask</td>
			<td style="width:232px;">Schott Duran</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2.7 mm glas beads</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">N032.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2-Propanole</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">9866.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">30 cm Luer-Lock Extension Tube</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Acetone</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5025.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Bio-Beads SM-2 Adsorbent</td>
			<td style="width:232px;">Bio-Rad</td>
			<td style="width:305px;">152-3920</td>
			<td>need to be activated before first use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:389px;">CaCl2 Dihydrat</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">HN04.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Calcein</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:305px;">C0875</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Chloroform reagent grade</td>
			<td style="width:232px;">VWR Chemicals</td>
			<td style="width:305px;">22711324</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:389px;">DOPEATTO390</td>
			<td style="width:232px;">ATTO-TEC</td>
			<td style="width:305px;">AD 390-165</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Ethanol absolute</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">32205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt Single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">460 60 51V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt-F single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">91 66 017V</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:389px;">Keck clips</td>
			<td style="width:232px;">Schott</td>
			<td style="width:305px;">KC29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">L-&#945;-Phosphatidylcholine, 20% (Soy)</td>
			<td style="width:232px;">Avanti Polar Lipids</td>
			<td style="width:305px;">5416016</td>
			<td>store under inert gas at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NaCl 99.5% p.a.</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">3957.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanopore E100 wafer/chips</td>
			<td style="width:232px;">Micromotive (Mainz/Germany)</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nucleopore Track-Etch Membrane 0.4 &#181;m</td>
			<td style="width:232px;">Whatman</td>
			<td style="width:305px;">800282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oregon Green Dextran 488 (70 kDa)</td>
			<td style="width:232px;">life Technologies</td>
			<td style="width:305px;">D-7173</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oy647</td>
			<td style="width:232px;">Luminartis (M&#252;nster/Germany)</td>
			<td style="width:305px;">OY-647-T-1mg</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Rotilabo-syringe filtration, unsterile, pore-size 0.22 &#181;m</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">P 818.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sephadex G-50</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">G5080</td>
			<td>column material for size exclusion chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Silastic MDX4-4210</td>
			<td style="width:232px;">Dow Corning</td>
			<td style="width:305px;"></td>
			<td>curing agent for chip fixation onto cover glass support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">sticky-Slide 8-well</td>
			<td style="width:232px;">ibidi</td>
			<td style="width:305px;">80828</td>
			<td>multi-well chamber for the mounting onto glass slides (chip holder)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Three-way stopcock blue</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744410001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Tris Pufferan 99.9% Ultra Quality</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5429.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Triton-X 100</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">6683.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Whatman 0.2 &#181;m cellulose nitrate membrane filter</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">NH69.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Name</td>
			<td style="width:232px;">Company</td>
			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Equipment</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">B&#252;chi 461 water bath</td>
			<td style="width:232px;">B&#252;chi</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">B&#252;chi Rotavapor RE 111</td>
			<td style="width:232px;">B&#252;chi</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Cary Eclipse Fluorescence Spectrophtometer&#160;</td>
			<td style="width:232px;">Varian</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">LiposoFast Mini Extruder</td>
			<td style="width:232px;">Avestin</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Membrane pump&#160;</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">15430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanosight Nanoparticle Tracking Microscope</td>
			<td style="width:232px;">Malvern / Nanosight</td>
			<td style="width:305px;">LM 14C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NyONE microscope</td>
			<td style="width:232px;">Synentec</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Pump control</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">CVC 2II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sonicator bath Sonorex RK100H</td>
			<td style="width:232px;">Brandelin electronic</td>
			<td style="width:305px;">31200001107477</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Vaccum pump RC5</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">1805400204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Water bath W13</td>
			<td style="width:232px;">Haake</td>
			<td style="width:305px;">002-9910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Plasma Cleaner PDC-37G</td>
			<td style="width:232px;">Harrick Plasma</td>
			<td style="width:305px;">PDC-37G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Name</td>
			<td style="width:232px;">Company</td>
			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Software</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">ImageJ</td>
			<td style="width:232px;">Open Source</td>
			<td style="width:305px;">http://imagej.nih.gov/ij/</td>
			<td>scientific image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NanoCalcFX</td>
			<td style="width:232px;">Freeware</td>
			<td style="width:305px;">http://sourceforge.net/projects/nanocalc/</td>
			<td>data analysis/evaluation software for massive transport kinetic datasets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NTA 2.3 Analytical Software</td>
			<td style="width:232px;">Nanosight</td>
			<td style="width:305px;"></td>
			<td>data acquisition and analysis software for nanoparticle tracking microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NTA 2.3 Temperature Comms</td>
			<td style="width:232px;">Nanosight</td>
			<td style="width:305px;"></td>
			<td>temperature controle software for nanoparticle tracking microscope</td>
		</tr>
	</tbody>
</table>

membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution

転位した基板は、非起電性である場合、単一のタンパク質レベルでの膜タンパク質の輸送はまだ、詳細な分析を忌避します。かなりの努力がこの分野で行われているが、膜トランスポーターの解析に必要な溶媒を含まない脂質二重層の技術と組み合わせて、自動化ハイスループットトランスポート分析を可能にする技術はまれです。トランスポーターのこのクラスは、しかし、細胞の恒常性において非常に重要なので、薬剤開発や新たな洞察を得るための方法論における重要な目標は必死に必要です。 ここで紹介する原稿は、単一のトランスポーター解像度で膜タンパク質媒介輸送プロセスの分析のための新規バイオチップの確立と取り扱いについて説明します。バイオチップは、その設計において非常に平行であり、工業用グレードおよび量で製造することができるナノポアで囲まれたマイクロキャビティから構成されています。プロテイン保有するリポソームは、直接に適用することができますSSM-技術（固体担持脂質膜）を用いた自己組織化細孔スパニング脂質二重層を形成するチップ表面。膜のポア貫通部分は、中またはリアルタイムでマルチスペクトル蛍光読み出しを続けることができ、キャビティ空間のうち基板転位のためのインターフェースを提供し、自立しています。標準作業手順書（SOP）の設立は、機能的に再構成することができる事実上すべての膜タンパク質のチップ表面上のタンパク質-保有脂質二重層の簡単な設置を可能にします。唯一の前提条件は、非起電輸送基質用の蛍光読み出しシステムの確立で​​す。 ハイコンテンツスクリーニング用途は、並行して複数のチップを記録し、自動化倒立蛍光顕微鏡を使用することによって達成できるです。大きなデータセットを自由に利用できるカスタム設計解析ソフトウェアを用いて分析することができます。三色のマルチスペクトル蛍光読み出し、さらに、偽陽性の結果を排除し、別のイベントクラスに公平なデータ判別を可能にします。 チップ技術は、現在のSiO 2表面に基づいているが、金でコーティングされたチップ表面を使用してさらに官能化することも可能です。

ポア貫通膜は容易に自己集合したようにナノ構造チップ表面上に作成することができます。しかし、根本的なプロセスはまだ繊細でリポソームのサイズ、リポソーム集団の単分散性、ラメラ、脂質や塩分濃度と化学的表面特性のような多くのパラメータの影響を受けています。これらのパラメータのほとんどは、慎重に特徴付けられ、上記のプロトコルで標準化され?...

Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

単一のタンパク質解像度で高並列ナノ細孔チップシステムにより分析膜輸送プロセス

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

membrane-transport-processes-analyzed-highly-parallel-nanopore-chip

Research

JoVE Journal

Biology

11.6K ビュー.  Goethe University Frankfurt. The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

ビデオ: Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

リバーシブルビオチン化によって原形質膜タンパク質エンドサイトーシス率を測定

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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.

核輸送の1分子イメージング

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

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

大気圧質量分析法による単一細胞の直接分析

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

血漿レチノール結合タンパク質の膜受容体によってレチノール交通のリアルタイム解析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF顕微鏡を経由してシングルイベント解像度のGタンパク質共役型受容体のクラスリン依存性エンドサイトーシスの可視化

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBICプロトコルは、全マウントスキン準備中の単一細胞の解像度でのタンパク質発現を可視化

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

3Dで栽培ネイティブ腸とのCaco-2細胞において上皮輸送タンパク質の機能および発現を研究するための方法

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...