この出版につながる仕事はジョージア番号246550の下でマラガ大学と欧州連合（EU）7 番目のフレームワークプログラム（FP7/2007-2013）によって協調融資マリーキュリーCOFUNDプログラム「U-モビリティ」から資金提供を受けています。私たちの研究室での作業はVCにNIH、米国農務省/ NIFA、NSF、バードとBSFからの補助金によってサポートされています

1。タンパク質の発現<ol><li> 発現系の選択 システムを選択し、すなわち、ベクターおよび特定のモデル生物/細胞における目的タンパク質の発現のために最適なベクター送達方法。我々のアッセイは、最高の多数の細胞の一過性形質転換することにより達成される簡単に検出可能な量で試験したタンパク質の発現を必要とすることに注意してください。植物では、例えば、これは、最高の発現ベクターおよび送達系としてのアグロバクテリウムバイナリーとしてプラスミドを用いて達成される。 </li><li> バイナリー発現ベクターの構築 単独で又はエピトープタグに翻訳融合のいずれかで発現ベクターに関心対象のタンパク質のコード配列（単数または複数）をクローニングする。使用は、選択した発現系および遺伝子クローニングのための標準的な分子生物学的手順に適したベクター。アグロバクテリウム媒介遺伝子送達のために、スタンダを使用しても、バイナリーベクターを採用し、それらを導入このような植物組織のその後の接種EHA105、アグロバクテリウム株にRDプロトコル、。 </li><li> 植物種の選択 目的のタンパク質（単数または複数）発現される植物種を選択する。アグロバクテリウム媒介形質転換を受けやすい任意の植物種を用いることができるが、なお、選択された我々の植物種はアグロバクテリウム容易に影響を非常に受けやすく、成長させたベンサミアナタバコ 、であり、容易に接種する大きな葉を有する。 </li><li> 植物成長 長い一日の環境制御された条件（例えば、成長室）130（すなわち、16時間の下の鍋（19センチ×20センチ×20cm）に中のプロミックスBXと鍋に4から6週間の1の植物を育てる-150μEM-2、S-1 23℃での光と20°Cで8時間暗い）と40〜65パーセントの相対湿度。 時折、製造業者の指示に従って市販の製品と1.5.2肥やす。植物が成長されると、選択アグロバクテリウム接種のために（これらの長さの測定には葉柄が含まれていません）×70ミリメートル以上50ミリメートルの大きさに残します。 </li><li> アグロバクテリウムを接種 /適切な抗生物質（例えば、100mgを補充したYEP培地中で28°C（1％ペプトン、1％酵母エキス、0.5％NaCl）中で一晩、ステップ1.3から試験したタンパク質を発現するバイナリー構築物を保有するアグロバクテリウム株を成長させるLのストレプトマイシンおよびpPZP系- RSC2ベースのベクター24〜25）が10 mg / Lのリファンピシン。 1.6.2遠心浸潤緩衝液中でOD 600まで再懸濁した細胞、= 0.5 [10mMのMgCl 2、10mMのMesの液（pH 5.6）、100μMアセトシリンゴン]、そして室温で2時間インキュベートする。 1 mLの針なしの注射器を使用して、葉の背軸側に文化を浸透させる、植物に接続されている間葉は、その場で接種されることに注意してください。 1.6.3浸透後、16時間130〜150＆の光政権の下で72時間、植物を育てる＃181;のE、M-2、S-1収穫前に20℃で23℃/ 8時間の暗の光。 </li><li> プロテアソーム阻害剤の適用 テストされたタンパク質（S）は、プロテアソーム経路を介して分解されるという考えをサポートするために、軽減あるいは分解を遮断する必要のある、特定のプロテアソーム阻害剤MG132を使用し、28〜29をラクタシスチン。収穫前に4時間（ステップ2.1を参照）、それぞれの溶媒を用いて、10μMMG132（EMDミリポア）または10μMラクタシスチン（Sigma-Aldrich社）またはモック治療の葉にアグロバクテリウムを接種した葉の面積を浸透、すなわち、0.1％のDMSOまたは蒸留水。 </li></ol> 2。無細胞抽出物の調製<ol><li> 葉の収穫 葉の接種された領域、通常はCAを収穫。 200〜400新鮮重の物、そして液体窒素中で微粉末にそれらを挽く。代わりに、任意のビーズビーター（例えば、バイオスペック）または歯科混汞（例えば、TPC [詳細設定]を使用して、組織をビーズビート<html>直接分解緩衝液中の技術）（工程2.2を参照のこと）。 </li><li> タンパク質抽出 分解緩衝液500μLに接地組織を配置することによって、総タンパク質抽出物を調製[50mMのトリス-HCL（pH7.5）中、100mMのNaCl、10mMのMgCl 2、5mMのDTT、5mMのアデノシン5&#39;-三リン酸、および1×プロテアーゼ阻害剤カクテル（Sigma-Aldrich社）〕。プロテアーゼ阻害剤カクテルは、主に、セリン、システイン、アスパラギン、およびメタロに影響を及ぼし、26Sプロテアーゼと干渉しないことに留意されたい。 5分間12,000×gで2連続的な遠心分離によりエキスを明らかにする。 </li><li> タンパク質分解反応 チューブを微量遠心し、時間の期間を増加させるために、室温でそれらをインキュベートし、通常は20μL、抽出物の等容量を転送する。典型的には、サンプル時間ゼロ及び5、10、15、20、および30分の時点。 SDSゲル試料緩衝液中で煮沸することにより反応を停止し、ウェスタンブロッティングによってそれらを分析。 </li></ol>jove_title &quot;&gt; 3。イムノブロッティングによりタンパク質分解の検出<ol><li> ゲル電気泳動 3.1.1 SDS-ポリアクリルアミドゲル電気泳動によりタンパク質試料を解決し、標準的なプロトコル30に従ってニトロセルロース膜に分離したタンパク質をエレクトロ。 3.1.2 Bradford法（Bio-Rad）を用いてタンパク質濃度を決定し、レーンあたり総タンパク質50〜80μgのを読み込む。すべてのサンプルが均等にロードされていることを確認してください。ローディングコントロールの場合、このような50kDa付近の相対的な電気泳動移動度を有する主要なバンドとして移動推定のルビスコ大鎖のような遍在タンパク質種の強度を比較して、彼らは、クマシーブルー染色ゲル上またはポンソーS-または検出することができる蛍光SYPROルビー染色したニトロセルロース膜。 </li><li> ブロッキング 室温で1時間、TBST（10mMトリス-HCl、140mMのNaCl、0.05％のTween 20、pH7.4）中の5％スキムミルクで膜をブロックする。 </lI&gt; <li> 一次抗体 メーカーが推奨する濃度でTBST中の1％スキムミルクで一次抗エピトープ抗体を希釈し、穏やかに撹拌しながら4℃で、室温で1時間または一晩ブロックされた膜とインキュベートする。 </li><li> すすぎ 膜を一回15分間20mlのTBSTでリンスし、二回穏やかに攪拌しながら室温で5分間リンスされる。 </li><li> 二次抗体 製造業者により推奨されるようにTBST中の1％スキムミルクで西洋ワサビペルオキシダーゼ（HRP）とコンジュゲート二次抗体（例えば、抗ウサギIgG）を希釈し、穏やかに攪拌しながら室温で1時間、膜とともにインキュベートする。 </li><li> 検出 ステップ3.4で説明したように、再び膜を洗浄します。 TBSTで最後のすすぎの後、最も一般的にはECLキットを用い、HRP化学発光基質を用いて目的のタンパク質を可視化する。 </li></ol>

<ol>
	<li>Patton, E. E., Willems, A. R., Tyers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+control+in+ubiquitin-dependent+proteolysis:+don&#8217;t+Skp+the+F-box+hypothesis.">Combinatorial control in ubiquitin-dependent proteolysis: don&#8217;t Skp the F-box hypothesis.</a> Trends Genet. 14, 236-243 (1998).</li><li>Deshaies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCF+and+cullin/ring+H2-based+ubiquitin+ligases.">SCF and cullin/ring H2-based ubiquitin ligases.</a> Annu. Rev. Cell Biol. 15, 435-467 (1999).</li><li>Callis, J., Vierstra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+degradation+in+signaling.">Protein degradation in signaling.</a> Curr. Opin. Plant Biol. 3, 381-386 (2000).</li><li>Hellmann, H., Estelle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+development:+regulation+by+protein+degradation.">Plant development: regulation by protein degradation.</a> Science. 297, 793-797 (2002).</li><li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annu. Rev. Biochem. 67, 425-479 (1998).</li><li>Dharmasiri, S., Estelle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+regulated+protein+degradation+in+auxin+response.">The role of regulated protein degradation in auxin response.</a> Plant Mol. Biol. 49, 401-409 (2002).</li><li>Devoto, A., Muskett, P. R., Shirasu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ubiquitination+in+the+regulation+of+plant+defence+against+pathogens.">Role of ubiquitination in the regulation of plant defence against pathogens.</a> Curr. Opin. Plant Biol. 6, 307-311 (2003).</li><li>Itoh, H., Matsuoka, M., Steber, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+ubiquitin-26S-proteasome+pathway+in+gibberellin+signaling.">A role for the ubiquitin-26S-proteasome pathway in gibberellin signaling.</a> Trends Plant Sci. 8, 492-497 (2003).</li><li>Wang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+26S+proteasome+system+in+the+signaling+pathways+of+TGF-beta+superfamily.">The 26S proteasome system in the signaling pathways of TGF-beta superfamily.</a> Front. Biosci. 8, 1109-1127 (2003).</li><li>Pagano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+DNA+synthesis+and+mitosis+by+the+Skp2-+p27-Cdk1/2+axis.">Control of DNA synthesis and mitosis by the Skp2- p27-Cdk1/2 axis.</a> Mol. Cell. 14, 414-416 (2004).</li><li>Magori, S., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hijacking+of+the+host+SCF+ubiquitin+ligase+machinery+by+plant+pathogens.">Hijacking of the host SCF ubiquitin ligase machinery by plant pathogens.</a> Front. Plant Sci. 2, 87 (2011).</li><li>Gelvin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+plant+transformation:+the+biology+behind+the+&amp;#34;gene-jockeying&amp;#34;+tool.">Agrobacterium-mediated plant transformation: the biology behind the &amp;#34;gene-jockeying&amp;#34; tool.</a> Microbiol. Mol Biol. Rev. 67, 16-37 (2003).</li><li>Lacroix, B., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+bacterial+and+host+plant+factors+in+Agrobacterium-mediated+genetic+transformation.">The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation.</a> Int. J. Dev. Biol. 57, (2013).</li><li>Schrammeijer, B., 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=Interaction+of+the+virulence+protein+VirF+of+Agrobacterium+tumefaciens+with+plant+homologs+of+the+yeast+Skp1+protein.">Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein.</a> Curr. Biol. 11, 258-262 (2001).</li><li>Zaltsman, A., Krichevsky, A., Loyter, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+induces+expression+of+a+plant+host+F-box+protein+required+for+tumorigenicity.">Agrobacterium induces expression of a plant host F-box protein required for tumorigenicity.</a> Cell Host Microbe. 7, 197-209 (2010).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+targeted+proteolysis+in+plant+genetic+transformation+by+Agrobacterium.">Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium.</a> Nature. 431, 87-92 (2004).</li><li>Zaltsman, A., Lacroix, B., Gafni, Y., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+synthetic+Agrobacterium+T-DNA-protein+complexes+via+the+host+SCFVBF+ubiquitin-ligase+complex+pathway.">Disassembly of synthetic Agrobacterium T-DNA-protein complexes via the host SCFVBF ubiquitin-ligase complex pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 169-174 (2013).</li><li>Zhou, P., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+degradation+of+the+substrate+recognition+subunits+of+SCF+ubiquitin-protein+ligases.">Ubiquitination and degradation of the substrate recognition subunits of SCF ubiquitin-protein ligases.</a> Mol. Cell. 2, 571-580 (1998).</li><li>Galan, J. M., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-dependent+degradation+of+multiple+F-box+proteins+by+an+autocatalytic+mechanism.">Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 9124-9129 (1999).</li><li>Ayad, N. G., Rankin, S., Murakami, M., Jebanathirajah, J., Gygi, S., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tome-1,+a+trigger+of+mitotic+entry,+is+degraded+during+G1+via+the+APC.">Tome-1, a trigger of mitotic entry, is degraded during G1 via the APC.</a> Cell. 113, 101-113 (2003).</li><li>Guardavaccaro, D., 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=Control+of+meiotic+and+mitotic+progression+by+the+F+box+protein+b-Trcp1+in+vivo.">Control of meiotic and mitotic progression by the F box protein b-Trcp1 in vivo.</a> Dev. Cell. 4, 799-812 (2003).</li><li>Magori, S., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+counteracts+host-induced+degradation+of+its+F-box+protein+effector.">Agrobacterium counteracts host-induced degradation of its F-box protein effector.</a> Sci. Signal. 4, (2011).</li><li>Margottin-Goguet, F., Hsu, J. Y., Loktev, A., Hsieh, H. M., Reimann, J. D., Jackson, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophase+destruction+of+Emi1+by+the+SCFbTrCP/Slimb+ubiquitin+ligase+activates+the+anaphase+promoting+complex+to+allow+progression+beyond+prometaphase.">Prophase destruction of Emi1 by the SCFbTrCP/Slimb ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase.</a> Dev. Cell. 4, 813-826 (2003).</li><li>Tzfira, 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=pSAT+vectors:+a+modular+series+of+plasmids+for+fluorescent+protein+tagging+and+expression+of+multiple+genes+in+plants.">pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants.</a> Plant Mol. Biol. 57, 503-516 (2005).</li><li>Goderis, I. 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=A+set+of+modular+plant+transformation+vectors+allowing+flexible+insertion+of+up+to+six+expression+units.">A set of modular plant transformation vectors allowing flexible insertion of up to six expression units.</a> Plant Mol. Biol. 50, 17-27 (2002).</li><li>Lee, L. Y., Gelvin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-DNA+binary+vectors+and+systems.">T-DNA binary vectors and systems.</a> Plant Physiol. 146, 325-332 (2008).</li><li>Dafny-Yelin, M., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+multiple+transgenes+to+plant+cells.">Delivery of multiple transgenes to plant cells.</a> Plant Physiol. 145, 1118-1128 (2007).</li><li>Yang, P., 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=Purification+of+the+Arabidopsis+26+S+proteasome:+biochemical+and+molecular+analyses+revealed+the+presence+of+multiple+isoforms.">Purification of the Arabidopsis 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms.</a> J. Biol. Chem. 279, 6401-6413 (2004).</li><li>Fenteany, G., 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=Inhibition+of+proteasome+activities+and+subunit-specific+amino-terminal+threonine+modification+by+lactacystin.">Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin.</a> Science. 268, 726-731 (1995).</li><li>Ausobel, F. 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=.">.</a> Current Protocols in Molecular Biology. , (1987).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Kim, J. H., Kim, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+RING+E3+ubiquitin+ligase+AtAIRP3/LOG2+participates+in+positive+regulation+of+high+salt+and+drought+stress+responses).">The Arabidopsis RING E3 ubiquitin ligase AtAIRP3/LOG2 participates in positive regulation of high salt and drought stress responses).</a> Plant Physiol. 162, 1733-1749 (2013).</li></ol>

利害の対立が宣言されていません。

このアッセイは、植物組織における試験したタンパク質の発現に依存しているため、潜在的なプロテアソーム分解プロセスは、明らかに、生体組織内で既に発生した。我々は、最初の基準点となる時間ゼロサンプルのみで、抽出物中で、しかし、タンパク質の不安定化をアッセイする。したがって、我々は、無細胞アッセイとして定義します。 このアッセイの成功のた?...

ubiquitin/26Sプロテアソーム経路は、転写調節、細胞周期の進行およびシグナル伝達、ダウンレギュレーションまたはエンドサイトーシス受容体、とりわけ1-4を処理するなど、多様な生物学的反応を制御するための広範な機構として浮上している。この経路において、標的タンパク質は、第ユビキチン活性化酵素E1にチオールエステル結合を介して結合し、次いで、ユビキチン共役酵素E2のシステインアミノ酸残基に転残基をユビキチンによりタグ付けされ、最後に、E2は、ユビキチンリガーゼE3と相互作用する、タンパク質基質のユビキチン化をもたらす。最終的には、ポリユビキチン化タンパク質は、26Sプロテアソームによって認識され、分解される。このメカニズムでは、E3酵素は、基質を指定し、ubiquitin/26Sプロテアソームシステム（UPS）の重要な調節コンポーネントとして機能します。 E3リガーゼは、RINGドメインリガーゼとして、またはマルチサブSCF（Sの一環として、独立して機能することができ例えば、F-boxドメインリガーゼとしてkp1/Cullin/F-boxタンパク質）複合体。 SCF媒介性プロテアソーム分解経路は、転写、細胞周期、シグナル伝達5-10および他の多くの主要な細胞機能の調節に関与している。 細胞過程の調節に、これらの重要な役割に加えて、UPSは多くの植物 - 病原体相互作用の中心的舞台になります。例えば、証拠が増えアグロバクテリウムツメファシエンスを含むいくつかの植物病原体は、感染プロセス11を容易にするためのホストのUPSに依存していることを示唆している。アグロバクテリウムは、その自然のホストを表す植物に腫瘍性増殖を誘発し、それはまた、ヒトの細胞12,13に菌類1,2から、他の真核生物の広い範囲を変換することができます。その感染時に、アグロバクテリウムは、宿主細胞12月13日にDNAエレメント（T-DNA）およびいくつかの病原性（Virの）タンパク質をエクスポートします。これらのタンパク質の一つがVirF、最初に検出されたF-boxタンパク質である原核生物のゲノム14によって符号化される。 SCFユビキチンリガーゼ複合体、VirF、及びその機能宿主ホモログVBF 15の一部として、おそらくVirE2、それに付随する細菌および宿主のタンパク質から侵入する細菌のT-DNAの脱殻を容易にUPS媒介性タンパク質分解を介して 、アグロバクテリウム感染を容易にするとVIP1、それぞれ16,17。興味深いことに、VirFを含む多くのF-ボックスタンパク質は、原因自己ユビキチン化活性の18,19によってまたはF-ボックスタンパク質は、基質20〜23として役立つ可能性があるため、他のE3リガーゼによって媒介される、独自のタンパク質分解に本質的に不安定である。 F-ボックスタンパク質、他のユビキチンリガーゼ、および/またはそれらの基質の生化学的活性を研究する場合には、プロテアソーム分解のためのシンプルで信頼性の高いアッセイを使用することは非常に有用であろう。ここで我々は、細胞中のタンパク質の安定性を分析するためのそのようなプロトコルの1つを記載フリーシステム。このアッセイでは、UPS基材の安定性は、無細胞系において、例えば、F-boxタンパク質としてのプロテアソーム分解経路の必須成分の一つの存在下又は非存在下で分析される。一般に、我々は、植物組織中で試験タンパク質を発現するこれらの組織からの無細胞抽出物を調製し、ウェスタンブロッティングによって、目的のタンパク質（単数または複数）の量を監視する。タンパク質分解のUPS依存性機構は、特定のプロテアソーム阻害剤および/またはSCF成分、キュリンのドミナントネガティブ型の同時発現を使用して含めることによって実証される。我々は、F-boxタンパク質VBF 15によって、シロイヌナズナVIP1 17タンパク質のプロテアソーム分解を用いてこのアッセイを例示する一方で、それは他のプロテアソーム基質の安定性を調べるために使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="662">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Protein assay kit</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">500-0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Proteinase inhibitor cocktail&#160;</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mini-Protean system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">165-8000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Semi-dry western blotting SD electrotransfer system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">170-3940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Affinity Purified Rabbit Anti-Ha</td>
			<td style="width:129px;">icllab</td>
			<td>RHGT-45A-Z</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG Peroxidase Conjugate</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:119px;">31460</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">BioTrace, NT nitrocellulose transfer membrane</td>
			<td style="width:129px;">Pall Corportation</td>
			<td style="width:119px;">27377-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Immobilon western chemiluminescent HRP substrate</td>
			<td style="width:129px;">EMD Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MG132</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>474790-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Lactacystin</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td>L6785</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td>
			<td style="width:129px;">Pierce</td>
			<td style="width:119px;">35055</td>
			<td></td>
		</tr>
	</tbody>
</table>

assaying proteasomal degradation in a cell-free system in plants

タンパク質分解のためにユビキチン - プロテアソーム経路は、事実上すべての真核生物における細胞機能の広範囲の調節のための最も重要なメカニズムの一つとして浮上している。具体的には、植物において、ubiquitin/26Sプロテアソームシステム（UPS）は、タンパク質分解を調節し、免疫応答、発生およびプログラム細胞死のプロセスを含む広範囲の開発に大きく貢献する。また、増加する証拠は、 アグロバクテリウムなどの多数の植物病原体は、植物-病原体相互作用におけるUPSの重要性を強調し、効率的な感染のホストにUPSを利用することを示唆している。 UPSの基質特異性は、E1とE2と協調して作用するE3ユビキチンリガーゼによって達成されるそれらにユビキチン分子の鎖を結合することによって分解宛ての特定のタンパク質分子を認識し、マークするリガーゼ。 E3リガーゼの一つのクラスは、SCF（Skp1と/ Cです具体的にはUPS基質を認識し、F-boxタンパク質成分を介してユビキチン化のためにそれらを標的ウルリン/ F-ボックスタンパク質）錯体。関心対象の生物学的プロセスにおけるUPSの潜在的役割を調査するために、UPS媒介性タンパク質分解のためのシンプルで信頼性の高いアッセイを考案することが重要である。ここでは、植物、無細胞系を用いてそのようなアッセイを記載している。このアッセイは、F-boxタンパク質 - 基質相互作用に特に焦点を有する多様な細胞プロセスにおける調節タンパク質分解の役割の研究に適合させることができる。

Zaltsman ら 17から適応図1は 、無細胞系におけるプロテアソーム分解の検出のための代表的な実験を示す。具体的には、NにSCF VBF経路 を介して VBFのF-ボックスタンパク質による植物防御関連タンパク質VIP1の不安定化を実証ベンサミ 。シロイヌナズナVBFおよびHA-タグ化VIP1（HA-VIP1）タンパク質を一過性同時発現させ、発現した葉の抽出物中...

Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

Assaying Proteasomal Degradation in a Cell-free System in Plants

標的タンパク質分解は、細胞機能の主要な調節機構を表す。その後、26Sプロテアソームのために、分子「タグ」を提供する標的タンパク質にユビキチン鎖が付加し保存され、ユビキチン-プロテアソーム経路を介して行われます。ここでは、タンパク質のプロテアソーム分解のための、シンプルで信頼性の高い無細胞アッセイを記述します。

植物では無細胞系でプロテアソームによる分解をアッセイ

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

assaying-proteasomal-degradation-in-a-cell-free-system-in-plants

Research

JoVE Journal

Biology

14.3K ビュー.  Stony Brook University, State University of New York. 標的タンパク質分解は、細胞機能の主要な調節機構を表す。その後、26Sプロテアソームのために、分子「タグ」を提供する標的タンパク質にユビキチン鎖が付加し保存され、ユビキチン-プロテアソーム経路を介して行われます。ここでは、タンパク質のプロテアソーム分解のための、シンプルで信頼性の高い無細胞アッセイを記述します。 

ビデオ: Assaying Proteasomal Degradation in a Cell-free System in Plants

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

無細胞培地でボルバキアの維持

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

生物学

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2のキナーゼ活性を測定する in vitroで</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

実装するためのプロトコル<em&gt;エシェリヒア·コリ</em合成生物学のための&gt;に基づくTX-TLの無細胞発現系

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

β-カテニンの分解にの再構成<em&gt;アフリカツメガエル</em&gt;卵抽出

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

一チャネルセル付パッチクランプ法

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

植物では無細胞系でプロテアソームによる分解をアッセイ

要約

さらに動画を探す

この動画の章

無細胞培地でボルバキアの維持

パイロシーケンシング：正確なジェノタイピングのための簡単な方法

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス

LRRK2のキナーゼ活性を測定する in vitroで

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

実装するためのプロトコル

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

β-カテニンの分解にの再構成

一チャネルセル付パッチクランプ法

植物では無細胞系でプロテアソームによる分解をアッセイ

要約

さらに動画を探す

この動画の章

無細胞培地でボルバキアの維持

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス

LRRK2のキナーゼ活性を測定する in vitroで

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

実装するためのプロトコル

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

β-カテニンの分解にの再構成

一チャネルセル付パッチクランプ法

パイロシーケンシング：正確なジェノタイピングのための簡単な方法