この作品は、イノベーションのためのカナダ基金（CFI）、健康研究（CIHR）のカナダの研究所、およびカナダ自然科学工学研究評議会（NSERC）からの補助金によって支えられている。

パート1：インフルエンザの破壊ビリオン<ol><li>ベックマンオプティマで使用するためのTLA - 120.2ローターに収まるように設計されたものベックマンポリカーボネート遠心管（11ミリメートル× 34ミリメートル）にMNTバッファー750μlの（20mMのMES、150mMのNaCl、30mMトリス、pH 7.5）を追加するマックス- Eの超遠心機。 </li><li>そのチューブに、インフルエンザウイルス（2 mg / mlのH3N2 X - 31 A/AICHI/68株）500μlを添加します。数回上下にピペッティングしてMNTバッファを使ってウイルスを混ぜる。 </li><li> TLA - 120.2ローターを使用してベックマンオプティママックス- Eの遠心機で、109000 × gで、4℃で10分間遠心します。 </li><li>上清を除去し、500μlの混乱のバッファーでペレットを再懸濁さは（100mMのKCl、5 mMのMgCl 2、5％（w / v）のグリセロール、50 mMのオクチルグルコシド、10 mg / mlのリゾレシチン、1.5mMのジチオトレイトール、100mMのMES、 pHは5.5）。 </li><li>渦は精力的にし、エッペンドルフのサーモミキサーで20分間31℃で振とうする。 </li></ol>パート2：グリセロール勾配土砂速度遠心<ol><li> 1 mlの70％（v / v）グリセロール、0.75ミリリットル、50％グリセロール、0.375ミリリットル40％グリセロール、および1.8：ベックマンultraclearの遠心管（13ミリメートル× 51ミリメートル）にグリセロールの以下の量を配置することによって、グリセロール勾配を準備ミリリットル33％グリセロール。グリセロールのソリューションは、NMのバッファー（150mMのNaCl、50mMのMES、pH5.5）で純粋なグリセリンを混合して作られています。また、グリセロールとバッファの両方を含むバランスのチューブを準備する。 </li><li>渦再びウイルスのサンプルを破壊し、グリセロール勾配の上にそれを読み込みます。 </li><li>ベックマンMLS - 50スイングバケットローターで、217000 × gで、4℃で3.75時間のために勾配を遠心分離します。 </li><li>遠心分離が完了したら、手動でチューブの上からグラデーションの250μlのアリコートを収集する。 ℃の氷の上または4で分画してください</li></ol>パート3：SDSポリアクリルアミドゲル電気泳動法によりグリセロール勾配分画の分析<ol><li>各フラクションから新しいチューブに20μlのを削除します。 </li><li> 5x SDS - PAGEサンプルバッファー5μlを添加します。 </li><li> 95〜ヒート℃で5分間。 </li><li>簡単にサンプルをスピンダウンし、10％ポリアクリルアミドゲル上にロードし、井戸の一つで、分子量マーカーが含まれています。 </li><li>ブロモフェノールブルーローディング色素がゲルの底部近くに達するまでゲルにサンプルを実行します。 </li><li>クーマシーブルーでゲルを染色する。 </li></ol>パート4：vRNP画分の濃縮<ol><li>主にNPを含むグリセロール画分を選択し、それらを組み合わせて、二ベックマンポリカーボネート遠心管（11ミリメートル× 34ミリメートル）にそれらを配布する。 </li><li>上下ジエチルピロカーボネート（DEPC）処理した超純水、及びピペットで混合するために数回、各チューブを埋める。 </li><li>ベックマンTLA - 120.2ローターで、157000 × gで、4℃で4.5時間遠心する。 </li><li>上清を除き、DEPC処理水50μlにペレットを再懸濁します。 </li><li>必要に応じて、濃縮されたvRNPsの280は、測定することができます。 NPは、プールされた画分の主要コンポーネントの存在として、NPのモル濃度の推定値は55350 M -1 cm -1のその吸光係数を（ProtParam 3を介して決定される）を使用して計算することができます。 </li><li>一定分量精製vRNPsを、そしてそれらを° C、後で使用するために-80℃で凍結保存する。 </li></ol>パート5：vRNPsのネガティブ染色<ol><li>たてのとフィルタリングの2倍MNT緩衝液（20mM MES、150mMのNaCl、30mMトリス、pH 7.5）を9μLに精製されたvRNPの1μlを希釈します。他のバッファは、vRNPsを希釈すると試料のグリッド（ステップ5.5）洗浄するためにOKですが、酢酸ウラニルが負の染色に使用されている場合PBSを使用しないでください。 </li><li>以前に30秒間parlodionと炭素膜で被覆試験片（銅TEM）グリッドをグロー放電。 </li><li>ピンセットでたてのグロー放電試験片のグリッドを保​​持し、試料のグリッド上に希釈vRNPの5μlをドロップして適用されます。 </li><li> 8分のグリッド上で試料の滴を残す。 </li><li>待っている間、ベンチにパラフィルムの小さなストリップを配置し、そして10μlのMNTバッファの各（グリッドを洗浄する）、そしてたて染色液の80μlをドロップ（1％モリブデン酸アンモニウムまたは1を含む2滴を分注するパラフィルム上に％の酢酸ウラニル）。 </li><li>吸着の8分後、ろ紙の切れ端で試料のグリッド（三角にカット）からサンプル溶液の一部を放出する。試料グリッドを乾燥させてはいけない。 </li><li> 1分の合計時間のために10μlのMNTバッファの各々を含む2滴の試料の格子を洗います。これは、慎重にドロップで試料の格子を下げるし、ろ紙の切れ端で試料の格子（試料のグリッドは、乾燥させることなく）からサンプル溶液の一部をオフにウィッキングすることによって行われます。 </li><li>直ちに試料のグリッドからバッファの最後の一滴をオフウィッキング後に、完全なLYの水没は、標本の染色液滴内のグリッド、1分間待ちます。 </li><li>完全にろ紙片を持つ試料の格子から染色液をオフに放出する。 </li><li>透過型電子顕微鏡下で観察する前に、数分間空気乾燥して試料のグリッドを許可する。 </li></ol>パート6：代表的な結果： <img src="/files/ftp_upload/old/1105_2009_1_22_1/1105_Figure2.png" alt="図2" width="600" height="215" />図1 インフルエンザNP（〜56 kDa）は、ウイルスリボ核タンパク質複合体で見つかった主なタンパク質は、NPのバンドは、一般的にvRNPs（図1）を含有する画分では最強のバンドです。 NPに加えて、それぞれのインフルエンザのvRNPは、複雑な（82、86及び86.5 kDaの分子量）三量体RNAポリメラーゼのコピーが含まれています。彼らの豊かさがNPのそれに比べて低いので、これらは、またはクーマシーブルー染色により表示されない場合があります。ゲルの銀染色が行われ、代わりにCommassie青の汚れ、場合ポリメラーゼは、しかし、検出することができます。インフルエンザマトリックスタンパク質M1（〜28 kDa）はNP蛋白質のピーク分画が存在する分画で最小限に存在する必要があります。 <img src="/files/ftp_upload/old/1105_2009_1_22_2/1105_3.png" alt="図3" width="600" height="360" />図2 透過型電子顕微鏡下で可視化などの負染色vRNPsの長さは120nmの（図2a）に約30nmである任意の長さを持つロッド状の粒子に似たvRNPsをもたらす必要があります。オリゴマーのNPがさらに二重らせんリピート構造に折り畳まれているNPの分子の鎖のように構成されているので、これらの棒状粒子のいずれかの端にループし、時には（図2b）見ることができます。

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> Virology. 202, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> J Mol Biol. 374, 910-916 (2007).</li><li>Gasteiger, E., Walker, J. 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> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> Virol J. 4, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> Biophys J. 87, 2749-2758 (2004).</li></ol>

vRNPsの精製がKemler ら （1994）によって記載された手順に基づいている。1私達と他の人も自分の核移入を勉強するvRNPsを分離するためにこのプロトコルを使用している。2,4,5 我々は、ウイルスゲノムはRNAで構成されているためRNaseフリーヒントやvRNPsを操作するチューブの使用をお勧めします、そのためRNAの存在下で容易に分解する。さらに、すべて?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>H3N2 X-31 A/AICHI/68 influenza A</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>490715</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-8250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G33-1</td>
<td>&nbsp;</td>
<tr>
<td>Octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O-8001</td>
<td>&nbsp;</td>
<tr>
<td>Lysolecithin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4129</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9779</td>
<td>&nbsp;</td>
<tr>
<td>Coomassie brilliant blue G-250</td>
<td>&nbsp;</td>
<td>Kodak</td>
<td>1367796</td>
<td>&nbsp;</td>
<tr>
<td>diethyl pyrocarbonate (DEPC)-treated water</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>750024</td>
<td>&nbsp;</td>
<tr>
<td>Uranyl Acetate</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>19481</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Molybdate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A-674</td>
<td>&nbsp;</td>
<tr>
<td>Optima MAX-E Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>434491</td>
<td>&nbsp;</td>
<tr>
<td>MLS-50 Rotor Package, Swinging Bucket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>367280</td>
<td>&nbsp;</td>
<tr>
<td>TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362046</td>
<td>&nbsp;</td>
<tr>
<td>Eppendorf Thermomixer</td>
<td>&nbsp;</td>
<td>Brinkman</td>
<td>022670000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification and visualization of influenza a viral ribonucleoprotein complexes

インフルエンザウイルスゲノムは8つの負のセンス、個々にインフルエンザの複数のコピーウイルスribonulceoprotein粒子（vRNPs）に核蛋白（NP）が充填された一本鎖RNA分子で構成されています。インフルエンザvRNPsは、ウイルスエンベロープで囲まれています。セルの入力時に、しかし、これらのvRNP複合体は、それらが宿主核輸送機構へのアクセスを細胞質に放出されています。インフルエンザvRNPsとインフルエンザのゲノムの複製の核移入を研究するために、それはウイルスの他のコンポーネントがこれらのプロセスに干渉しないように隔離されたvRNPsで作業するのに便利です。ここで、我々は、インフルエンザウイルスからこれらvRNPsを精製する手順を説明します。手順は、包まれたビリオンからvRNP複合体を解放するために、インフルエンザの混乱洗剤とビリオンから始まります。 vRNPsは、インフルエンザ速度沈降によって33から70パーセント不連続グリセロール勾配でビリオンの他のコンポーネントから分離されています。グリセロール勾配から得られた分画は、クマシーブルーで染色後を経由してSDS - PAGEで分析されています。 NPを含有するピーク画分を一緒にプールし、遠心分離により濃縮されています。濃縮後、vRNPsの整合性は、ネガティブ染色した後、透過型電子顕微鏡によるvRNPsの可視化によって検証されます。グリセロール密度勾配による精製はKemlerからその修正したものです<em&gt;ら。</em&gt;（1994）<sup&gt; 1</sup&gt;、および負の染色が呉で行われている<em&gt;ら。</em&gt;（2007）。<sup&gt; 2</sup

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

インフルエンザウイルスのゲノムは、RNAとタンパク質の8つの独立した複合体と呼ばれる、ウイルスのリボ核タンパク質複合体（vRNPs）で構成されています。本論文では、グリセロール勾配精製とインフルエンザvRNPsの透過型電子顕微鏡の可視化について説明します。

インフルエンザウイルスリボ核タンパク質複合体の精製と可視化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

purification-visualization-influenza-viral-ribonucleoprotein

Research

JoVE Journal

Biology

12.9K ビュー.  University of British Columbia - UBC. インフルエンザウイルスのゲノムは、RNAとタンパク質の8つの独立した複合体と呼ばれる、ウイルスのリボ核タンパク質複合体（vRNPs）で構成されています。本論文では、グリセロール勾配精製とインフルエンザvRNPsの透過型電子顕微鏡の可視化について説明します。

ビデオ: Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

生物学

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

磁気共鳴イメージングのための準備、精製、および造影剤として使用するための希土類錯体のキャラクタリゼーション

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104の精製、タンパク質Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

RIP-チップを用いた細胞抽出物からmRNAを、マイクロRNAとリボ核タンパク質複合体のタンパク質成分の分離と​​同定するための方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

転写産物および代謝物からの精製<em&gt;ショウジョウバエ</em&gt;ヘッド

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

細胞壁の弾性特性のAFMベースのマッピング：ティッシュ、携帯電話、および細胞内の解像度で

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

組織解離と差動濾過により移植のためにミトコンドリアの迅速な単離および精製

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

インフルエンザウイルスリボ核タンパク質複合体の精製と可視化

要約

さらに動画を探す

この動画の章

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

磁気共鳴イメージングのための準備、精製、および造影剤として使用するための希土類錯体のキャラクタリゼーション

Hsp104の精製、タンパク質Disaggregase

高活性酵母のリボソームのクロマトグラフィー精製

RIP-チップを用いた細胞抽出物からmRNAを、マイクロRNAとリボ核タンパク質複合体のタンパク質成分の分離と同定するための方法

のタンパク質複合体の同定

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

転写産物および代謝物からの精製

細胞壁の弾性特性のAFMベースのマッピング：ティッシュ、携帯電話、および細胞内の解像度で

組織解離と差動濾過により移植のためにミトコンドリアの迅速な単離および精製

インフルエンザウイルスリボ核タンパク質複合体の精製と可視化

要約

さらに動画を探す

この動画の章

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

磁気共鳴イメージングのための準備、精製、および造影剤として使用するための希土類錯体のキャラクタリゼーション

Hsp104の精製、タンパク質Disaggregase

高活性酵母のリボソームのクロマトグラフィー精製

RIP-チップを用いた細胞抽出物からmRNAを、マイクロRNAとリボ核タンパク質複合体のタンパク質成分の分離と​​同定するための方法

のタンパク質複合体の同定

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

転写産物および代謝物からの精製

細胞壁の弾性特性のAFMベースのマッピング：ティッシュ、携帯電話、および細胞内の解像度で

組織解離と差動濾過により移植のためにミトコンドリアの迅速な単離および精製

RIP-チップを用いた細胞抽出物からmRNAを、マイクロRNAとリボ核タンパク質複合体のタンパク質成分の分離と同定するための方法