我々は、このプロトコルの開発と援助のためにサイモンアルベルティに感謝。この作品は、国立衛生研究所（GM25874）、ハワードヒューズ医学研究所のInvestigatorship（SLまで）、および国立科学財団博士号を取得する前の訓練助成金（RHまで）からの助成金によって支えられて。

<html>パート1：ゲルの準備<ol><li>ゲルキャスティングトレイを組み立てます。水平方向のDNAの電気泳動のための標準機器を使用することができます。大量のサンプルでは、​​最大4つの50ウェルコームを持つx 24センチメートルスラブ20cmの準備。これらは、ブロットの画像を歪ませることができるようにスラブが、傷を持っていないことを確認してください。 </li><li> 1X TAEの1.5％アガロース溶液（培地または高ゲル強度、低いEEO）を作成します。ボリュームは完全に水没するのに十分な櫛歯でなければなりません - あなたは、検出を最大化するために、できるだけ多くのサンプルを読み込むことになるでしょう。アガロースが完全に溶解するまで電子レンジで混合。 </li><li>急速に10％の株式を0.1％にSDSを追加。ミックスに渦巻く。いくつかのアガロースがこのステップ中に固化する場合、ホットプレートを用いて再溶解し、沸騰を避けるために注意してください。 </li><li>キャスティングトレイにソリューションを注ぐ。彼らは後に転送を妨げる可能性があるので、あらゆる気泡アウト熊手に櫛を使用してください。 </li><li>ゲルが設定された後、櫛を削除し、ゲルのタンクにゲルを置く。完全に水没1X TAEのゲルは、0.1％SDSを含む。 </li></ol>第2部：準備のサンプル<ol><li>酵母溶解物のハイスループット分析のために、我々は、96ウェルブロック単位で急速に撹拌しながら一晩生育2 mlの培養液から始まります。この場合、それぞれの文化が過剰発現は、目的のタンパク質です。低濃度のタンパク質を分析する際に、大きな文化のボリュームを使用する必要があります</li><li>室温で5分間2000 RCFで遠心分離により細胞を回収。 </li><li>再び水と遠心で細胞を再懸濁します。 </li><li> 1ミリリットルspheroplasting溶液中に再懸濁します。 30 ° C（あなたが顕微鏡によりspheroplasting効率を確認すること）で約30分間インキュベートする。 </li><li>室温で5分間、800 RCFで遠心する。完全に上清を取り除く。 </li><li> 100 mlの溶解バッファーでペレットスフェロプラストを再懸濁します。 </li><li>テープと2分間高速でボルテックスでブロックをカバー。 </li><li> 2分は4000 RCFでペレットを細胞破片。 </li><li>慎重に新しい容器、例えば、96ウェルPCRプレートに上清を取り除く。 </li><li>必要に応じて、溶解液のタンパク質濃度を決定する。 </li><li> 1Xサンプルバッファーを含む溶解液を生成するためにサンプルに4Xサンプルバッファーを添加します。室温で5分間インキュベートする。 </li><li>ゲルをロードします。必要に応じて、サンプルの量の半分を保存し、SDS - PAGE分析のためにそれを煮る。後で転送の程度を監視するために、事前に染色したSDS - PAGEのマーカーが含まれています。さらに、非常に大規模なタンパク質（例えば、ニワトリ胸筋エキス）から成る分子量マーカーを解決複合体の大きさを推定するために使用することができます。 </li><li>色素フロントがゲルの端から約1センチメートルに達するまで、低電圧（≤3 V / cmのゲルの長さ）で動作します。これには数時間かかります。ゲルは涼しいままであることが重要です、それ以外の低分子量のタンパク質（例えば、単量体）の拡散は、それらの検出を制限することができます。 </li></ol>パート3：転送<ol><li>ゲルと同じ大きさにニトロセルロースの部分をカット。 </li><li>ゲルと同じ大きさにGB002吸い取り紙のGB004と8個、20個をカット。芯として使用されるGB004の追加部分をカットし、ゲルよりも約20cm広いそれを作る。 </li><li>ニトロセルロース、芯、および1X TBSでGB002の4個を浸し。 </li><li>ドライGB004の20個、その後乾いたGB002の4枚、その後プリウェットGB002の一枚：プラスチック容器では、次のように紙のスタックを組み立てる。ニトロセルロースは、このスタックの一番上に置く。 </li><li>過剰な実行中のバッファを削除するには、水で簡単にキャスティングトレイ上でゲルをすすぐ。その後、慎重にスタックにプッシュトレイからゲルをスライドし始める。トレイからゲルをスライドさせながら、必要に応じてTBSで膜を消す。余分なバッファは、ゲルの下敷きになってから泡を防ぐのに役立ちます。転送ピペットは、この目的に適しています。 </li><li>ゲルがスタックに移動された後、泡が十分に確認してください。いずれかが存在する場合、ゲルの端を持ち上げ、気泡が正常に機能できるようになるまでバッファを再適用します。 </li><li>ゲルの上に残りの3つのプレ接液GB002ピースを置く。スタックの上部にしっかりとピペットを圧延して、すべてのレイヤーの間に徹底的な連絡を確保する。 </li><li> TBSを含む二つの高架トレイと脇腹転送スタックを。芯のどちらかの端がTBSに水没するようなスタック全体でプリウェット芯を羽織る。 </li><li>余分な重量を（例えば、水の500mlボトル）を有する追加のプラスチック製のトレイでアセンブル転送スタックをカバーする。 </li><li>転送は3時間の最小値、または一晩のために続行することができます。 </li><li>転送後、メンブレンは標準ウェスタンブロット法で処理することができます。 </li></ol> Spheroplastingソリューション 1.2 M D -ソルビトール 0.5mMのMgCl 2の 20mMトリス、pH7.5の 50mMのBME（新鮮な追加） は0.5 mg / mlのザイモリアーゼ100T（新鮮な追加） coration：下線;&quot;&gt;溶解バッファー 100mMのトリス7.5 50mMのNaCl 10mMのBME（新鮮な追加） プロテアーゼ阻害剤（新鮮な追加） 4Xサンプルバッファー 2X TAE 20％のグリセロール 8％SDS 好みにブロモフェノールブルー

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. 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=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. 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=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD - AGEは、最初Kryndushkinらによって報告された。1のSDS耐性の複合体を研究するために[PSI +]酵母におけるプリオン、と以来、プリオンと非プリオン凝集体2から9の両方を研究する広範な使用を発見した。しかし、アガロースゲルの膜以下の電気泳動のタンパク質の転送には問題がある、と歪んだブロットの画像5で発生する可能性があります。また、最も一般的に使用される水中エレクトロブロッティングの手法は、このようにゲルの大きさと処理できるサンプル数のための実際的な制限が導入されています。我々は、下向きにキャピラリートランスファー10、ゲルからニトロセルロース膜にタンパク質を転送するためにドライブロッティング紙のスタックを使用する単純な手順を採用することにより、これらの問題に対処している。キャピラリートランスファーには、歪みを防止し、大きなゲルを容易に処理することが可能になります。 SDD - AGEを使用する前に、次の事項を考慮する必要があります。原油サンプル（例えば、溶解液）の場合は、特定のタンパク質の免疫検出が必要です。 SDD - AGEは完全に興味の蛋白質複合体を変性させないので、検出される蛋白質（s）はアミロイド地域の外にエピトープタグを負担しなければならない。彼らは通常のSDS - PAGEのためになるように溶解液は、一般的に2つの重要な違いで、調製することができる。最初に、タンパク質分解による劣化を防止するために増加に注意する必要があります。ここで使用される部分的に変性条件下では、プロテアーゼを不活性化するには不十分であり、また、タンパク質分解の標的蛋白質が影響を受けやすくすることができます。少なくとも2倍の推奨濃度で完全なプロテアーゼ阻害剤のカクテルを使用してください。第二に、サンプルを加熱することは避けるべきです。全単量体陰性コントロールが必要な場合には、例えば高分子量の種が単量体蛋白質に最もアミロイドを復元する共有結合修飾、95℃で10分間インキュベート° Cを使用することができる、によるものではないことを確認する。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
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screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

アミロイド凝集は、下等生物に有益な役割を持っていると考えられているコンホメーション、自己鋳型タンパク質である多数のタンパク質のミスフォールディングの病態と根底プリオンの感染特性、関連付けられています。アミロイドは、不溶性と構造の異質性のために勉強するのが困難でした。しかし、大きさと界面活性剤不溶性に基づくアミロイド重合体の分解能は、洗剤、アガロースゲル電気泳動（SDD - AGE）セミ変性によって可能とされています。この手法は、アミロイドコンフォメーションの変種の検出と特性評価のための広範な使用を見つけることです。ここで、我々はそのような新規のプリオンおよび他のアミロイド蛋白質のためのスクリーンのような大規模なアプリケーションでその使用を容易にこの技術の適応を示しています。新しいSDD - AGEの方法は、より高い信頼性と使いやすさのためにキャピラリートランスファーを使用して、任意のサイズのゲルを収容することができます。従って、細胞または精製タンパク質から調製したサンプルの多くは、タグ融合タンパク質のSDS不溶性配座の存在を同時に処理することができます。

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

SDD - AGEは細胞内のポリマーのようなアミロイドの検出と特性評価のための有用なテクニックです。ここでは、大規模なアプリケーションにこのテクニックは、従順になるの適応を示しています。

セミ変性洗剤-アガロースゲル電気泳動によりアミロイド凝集のためにスクリーニング

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Research

JoVE Journal

Biology

26.7K ビュー.  Whitehead Institute for Biomedical Research. SDD - AGEは細胞内のポリマーのようなアミロイドの検出と特性評価のための有用なテクニックです。ここでは、大規模なアプリケーションにこのテクニックは、従順になるの適応を示しています。

ビデオ: Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

変性剤濃度勾配ゲル電気泳動（DGGE）

生物学

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

変性尿素ポリアクリルアミドゲル電気泳動（尿素PAGE）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

細胞溶解物からの多タンパク質複合体の解析のための青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

商業カセットを再利用することでタンパク質ゲルを注ぐと実行

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

DNA断片の分離のためのアガロースゲル電気泳動

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

ムチンアガロースゲル電気泳動：高分子量の糖タンパク質のためのウェスタンブロッティング


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

線虫の加齢とともに固有の蛋白質の集合の変化を研究するためのメソッド

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共焦点顕微鏡を明らかに細胞表面受容体画像相関分光法による凝集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

ブルー ネイティブ ポリアクリルアミドゲル電気泳動法と免疫ブロットを使用して培養されたひと細胞のミトコンドリア呼吸鎖複合体の解析

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...