1。サンプル準備ウエスタンブロット法の最初のステップは、サンプルの準備です。ゲルの細胞や組織を実行するためのサンプルを準備するには、目的のタンパク質を解放するために溶解する必要があります。 <ul><li>汎用性と可溶性タンパク質の抽出のために使いやすい、便利、すぐに使用できる試薬はCytoBusterまたはPhosphoSafeです。これらの抽出バッファーには、哺乳類と昆虫細胞から可溶性タンパク質を効率的に抽出するために最適化された洗剤を含まれています。 CytoBusterのタンパク質抽出試薬の穏やかな、非イオン性組成物は、超音波処理や凍結/融解などの二次治療を必要とせずに機能的に活性な内因性または発現タンパク質の分離を可能にします。 PhoshoSafeは、4つのホスファターゼ阻害剤を含む付加的な利点を持っています。 <ol><li>低速の遠心分離（2500 xgで例えば5分）により細胞をペレット化は、よく細胞ペレットを排出。 </li><li> 10 6細胞（CytoBusterのタンパク質抽出試薬の最適量は、セルサイズに基づいて異なる場合があります）あたり150μLを使用してCytoBusterのタンパク質抽出試薬で細胞を再懸濁します。 </li><li> 5分間室温でインキュベートする。 </li><li>適切なチューブに移し、4℃で16000 × gで5分間スピン℃に</li><li>転送は、新しいチューブに（細胞抽出液）上清をクリアし、分析を進める。 </li></ol></li><li>特殊なタンパク質の抽出には、我々は高品質のプロテオキットを使用することをお勧めします。 EMDは、ミトコンドリアの分離、細胞内プロテオームの抽出、膜貫通タンパク質の抽出、および他の多くのためのダース以上のキットを持っています。詳細については、私たちのウエスタンブロットのランディングページをご覧ください。 </li><li> EMDはまた、プロテアーゼやホスファターゼ阻害剤のカクテルセットを幅広く販売しています。とすぐに溶解が発生すると、タンパク質分解、脱リン酸化し、変性が始まります。これらのイベントのサンプルは氷上または4℃で保管している場合は途方もなく遅くすることができます·すべての時間と適切な阻害剤でのCは、溶解バッファーに新鮮な追加されます。詳細については、私たちのウエスタンブロットのランディングページをご覧ください。 </li></ul> 2。 SDS - PAGE ウエスタンブロット法の第二段階は、タンパク質の分離です。タンパク質は分子量に基づいて分離されている。 <ul><li>あなたがあなた自身のPAGEゲルを作ることができる、しかし我々は市販のプレキャストゲルを使用することになります。 <ol><li>あなたのサンプルを準備した後、タンパク質濃度を決定する準備が整いました。 EMDの濃度を測定するには、このための2つのキットを持っており、一つは非干渉タンパク質アッセイキットおよびBCAタンパク質アッセイキットであることその他のであること。 </li><li>タンパク質濃度が評価されると我々は、ゲルをロードするための我々のサンプルを準備する準備が整いました。抗体は通常、目的のタンパク質の小さな部分（エピトープと呼ばれる）を認識し、このドメインは、タンパク質の立体構造内に存在していてもよい。この部分に対する抗体のアクセスを有効にするには、それはタンパク質を展開する必要があります。 </li><li>変性させ、アニオン変性界面活性剤ドデシル硫酸ナトリウム（SDS）とローディング緩衝液を使用し、5分間95〜100℃で混合物を沸騰。使いやすく、便利なローディングバッファーには、4Xサンプルバッファーです。 </li></ol></li><li>トレイルミックスのタンパク質マーカーとよく最初の車線をロードする。このマーカーは、電気泳動を監視できるようにする3つの基準バンドを持っています。ゲルを染色されると、10バンドは10から225 kDaのからまで表示されます。 </li><li>バッファを実行して電気泳動ユニットを埋める。 PAGEゲルの場合、これは通常1Xトリス - グリシンです。詳細なバッファのレシピの場合は、ウェスタンブロッティングのページをご覧ください。 EMDは、私たちのOmniPur化学ラインで高品質のバッファの試薬を提供しています。 1時間220Vでゲルを実行します。 </li><li>タンパク質が分離した後、蛋白質が均等にかつ均一に移行したことを確認するために、クマシーブルーでゲルを染色。 RAPIDstainのに使える準備ができている超高感度の染色です。いいえ脱色は必要ありません。 </li></ul> 3。タンパク質の転送電荷を持つタンパク質が電界のゲルを通過するように誘導することができるのと同じように、タンパク質はPVDFまたはニトロセルロースのいずれかである、メンブレン上にゲルから電気的なフィールドで転送することができます。 <ul><li>今日は、ウェットの転送を行います。ウェット転送では、ゲルとメンブレンはスポンジと紙の間に挟まれている（スポンジ/紙/ゲル/メンブレン/紙/スポンジ）とすべてがない気泡はゲルとメンブレンの間に形成されている確認した後、しっかりと一緒にクランプされています。サンドイッチは、電界が印加される転写バッファーに浸漬される。負に帯電したタンパク質は、正に帯電した電極に向かって移動するが、膜はそれらを停止し、それらを結合する、との継続からそれらを防ぐことができます。 </li><li>膜の2種類が用意されています：ニトロセルロースおよびPVDF。どちらも動きます。今日は、PVDFを使用することになります。 PVDF膜は、メタノに浸かっRequireの5分間氷冷転送バッファに続いて数分の場合はl、。ゲルはまた、氷冷転送バッファの数分間平衡化する必要があります。そうしないと、転送中にゲルを減少することもあります。 </li><li>ウェット転送用バッファは、20％メタノールで1Xトリス - グリシンです。詳細なバッファーの調製は、当社のウェブサイトで入手できます。 </li><li>転送が完了したら、それは決して、エアポケットに転送さえ確保するためにタンパク質を可視化することをお勧めします。これは簡単にステインレッドアラートウエスタンブロットで行うことができます。この染色は、に使用準備ができており、脱色は水で簡単に行うことができます。 </li></ul> 4。抗体およびアクセサリの試薬<ul><li>抗原にプロービングし、抗体を行う最初のステップは、メンブレンをブロックすることです。メンブレンをブロックすると、非特異的バックグラウンドの結合を防ぎます。 </li><li>無脂肪牛乳やBSAのどちらかをブロッキング溶液として使用することができます。しかし、リン酸化特異的抗体を使用するとき、それは高いバックグラウンドの原因となるので、ミルクを使用することは推奨されません。 </li><li> EMDは、準備される非哺乳類遮断剤とブロットQuickBlockerであるSeaBlock、、など、いくつかの高品質なブロッキング試薬は、ブロッキング剤の使いすぐに提供しています。我々はまた、高品質のBSA（フラクションV）を利用している。 <ol><li>穏やかに撹拌しながら室温で膜1時間インキュベートします。 </li><li>インキュベーション後、TBSTまたはPBSTで3回洗浄する。 TBSTまたはPBSTを作るのは簡単で便利な方法は（PBS TWEEN錠/ TBS TWEEN錠）私たちの錠剤を使用することです。 </li></ol></li><li>膜がブロックされたら、一次抗体とインキュベートする準備が整いました。 EMDは30年以上にわたって業界で最高の抗体の保証と例外的な抗体の包括的なポートフォリオを提供してきました。すべてのEMDの抗体を用いて、我々が100％リスクのない保証を提供することを忘れないでください。抗体を購入し、あなたが望むあらゆる種の特異性やアプリケーションのためにそれを使用してください。抗体は、あなたの満足に動作しない場合は、我々は、他の製品で使用される完全な信用を提供します。詳細については、私たちのウェスタンブロッティングのWebページをご覧ください。 <ol><li>今日、我々はSignalBoostの溶液1を使用して私たちの一次抗体をインキュベートされます。 SignalBoostは、信号が大幅に強化できるように偉大な製品です。単に解決方法1で一次抗体をインキュベートし、1時間または通常どおりインキュベートする。さらにブロッキング剤を追加する必要はありません。代わりに、ブロッキング剤としてBSAや脱脂粉乳を使用することができます。 </li><li> TBSTで洗浄する。我々は、既製溶解するTBSTのタブレットを使用してTBSTを行います。 </li><li> SignalBoostの溶液2を使用して、二次抗体をインキュベートする。 1時間をブロッキングバッファーで二次抗体をインキュベートする。 TBSTで再び数回膜を洗浄してください。 </li></ol></li></ul> 5。検出 HRPコンジュゲート二次抗体の場合、ECLは、伝統的に使用されます。私たちが提供する優秀なECL試薬はRapidStepです。 RapidStepの利点は、管腔またはエンハンサーのないミキシングが必要ないことです。単純に膜を数回スプレーし、X線フィルムを使用して開発する。 RapidStepは、基質の添加後2時間までに低ピクトグラムの感度だけでなく、発光光を提供しています。

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Renart, J., Reiser, J., Stark, G. R., R, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc Natl Acad Sci U S A. 76 (7), 3116-3120 (1979).</li><li>Burnette, W. N. '. W. e. s. t. e. r. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=blotting':+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate+polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li><li>Ambroz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+quantification+accuracy+for+Western+blots+Image+Analysis.">Improving quantification accuracy for Western blots Image Analysis.</a> , (2006).</li><li>Quan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species-mediated+endoplasmic+reticulum+stress+and+mitochondrial+dysfunction+contribute+to+cirsimartitin-induced+apoptosis+in+human+gallbladder+carcinoma+GBC-SD+cells.">Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to cirsimartitin-induced apoptosis in human gallbladder carcinoma GBC-SD cells.</a> Cancer Lett Article. , (2010).</li><li>O'Malley, D. e. r. v. i. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+colonic+corticotrophin-releasing+factor+receptors+in+the+maternally+separated+rat+model+of+irritable+bowel+syndrome:+Differential+effects+of+acute+psychological+and+physical+stressors.">Alterations in colonic corticotrophin-releasing factor receptors in the maternally separated rat model of irritable bowel syndrome: Differential effects of acute psychological and physical stressors.</a> Peptides. 31 (4), 662-670 (2010).</li></ol>

作家アンナEslamiとジェシールハンは、この記事で使用する試薬や器具を生産するEMDケミカルズ社によって採用されています。

このビデオプレゼンテーションでは、我々は、サンプル調製、SDS - PAGE、抗体、および検出とブロッキング/プロービング膜であるウエスタンブロット法の主な手順を強調している。プロセスの各ステップについては、適切なプロトコルと適切な試薬は、高品質な結果を達成するために使用する必要があります。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CytoBuster&trade; Protein Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71009</td>
<td><a href="http://www.emdbiosciences.com/cytobuster">www.emdbiosciences.com/cytobuster</a> <a href="http://www.merck4biosciences.com/cytobuster">www.merck4biosciences.com/cytobuster</a></td>
</tr>
<tr>
<td>PhosphoSafe&trade; Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71296</td>
<td><a href="http://www.emdbiosciences.com/phosphosafe">www.emdbiosciences.com/phosphosafe</a> <a href="http://www.merck4biosciences.com/phosphosafe">www.merck4biosciences.com/phosphosafe</a></td>
</tr>
<tr>
<td>Non-Interfering Protein Assay&trade; Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>488250</td>
<td><a href="http://www.emdbiosciences.com/noninterfering">www.emdbiosciences.com/noninterfering</a> <a href="http://www.merck4biosciences.com/noninterfering">www.merck4biosciences.com/noninterfering</a></td>
</tr>
<tr>
<td>BCA Protein Assay Kit </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71285</td>
<td><a href="http://www.emdbiosciences.com/BCA">www.emdbiosciences.com/BCA</a> <a href="http://www.merck4biosciences.com/BCA">www.merck4biosciences.com/BCA</a></td>
</tr>
<tr>
<td>4X SDS Sample Buffer </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70607</td>
<td><a href="http://www.emdbiosciences.com/4XSDS">www.emdbiosciences.com/4XSDS</a> <a href="http://www.merck4biosciences.com/4XSDS">www.merck4biosciences.com/4XSDS</a></td>
</tr>
<tr>
<td>Trail Mix&trade; Protein Markers</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70980</td>
<td><a href="http://www.emdbiosciences.com/trailmix">www.emdbiosciences.com/trailmix</a> <a href="http://www.merck4biosciences.com/trailmix">www.merck4biosciences.com/trailmix</a></td>
</tr>
<tr>
<td>ProteoExtract&trade; Protein Extraction Kits</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/proteoextract">www.emdbiosciences.com/proteoextract</a> <a href="http://www.merck4biosciences.com/proteoextract">www.merck4biosciences.com/proteoextract</a></td>
</tr>
<tr>
<td>OmniPur&reg; Products</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/omnipur">www.emdbiosciences.com/omnipur</a></td>
</tr>
<tr>
<td>RAPIDstain&trade; Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>553215</td>
<td><a href="http://www.emdbiosciences.com/rapidstain">www.emdbiosciences.com/rapidstain</a> <a href="http://www.merck4biosciences.com/rapidstain">www.merck4biosciences.com/rapidstain</a></td>
</tr>
<tr>
<td>RedAlert&trade; 10X Western Blot Stain</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71078</td>
<td><a href="http://www.emdbiosciences.com/redalert">www.emdbiosciences.com/redalert</a> <a href="http://www.merck4biosciences.com/redalert">www.merck4biosciences.com/redalert</a></td>
</tr>
<tr>
<td>SeaBlock&trade; Reagent, Salmon Plasma</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>558300</td>
<td><a href="http://www.emdbiosciences.com/seablock">www.emdbiosciences.com/seablock</a> <a href="http://www.merck4biosciences.com/seablock">www.merck4biosciences.com/seablock</a></td>
</tr>
<tr>
<td>BLOT-QuickBlocker Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>WB57</td>
<td><a href="http://www.emdbiosciences.com/quickblocker">www.emdbiosciences.com/quickblocker</a> <a href="http://www.merck4biosciences.com/quickblocker">www.merck4biosciences.com/quickblocker</a></td>
</tr>
<tr>
<td>Albumin, Bovine Serum, Fraction V</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>12660</td>
<td><a href="http://www.emdbiosciences.com/bovineserum">www.emdbiosciences.com/bovineserum</a> <a href="http://www.merck4biosciences.com/bovineserum">www.merck4biosciences.com/bovineserum</a></td>
</tr>
<tr>
<td>PBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524653</td>
<td><a href="http://www.emdbiosciences.com/PBST">www.emdbiosciences.com/PBST</a> <a href="http://www.merck4biosciences.com/PBST">www.merck4biosciences.com/PBST</a></td>
</tr>
<tr>
<td>TBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524753</td>
<td><a href="http://www.emdbiosciences.com/TBST">www.emdbiosciences.com/TBST</a> <a href="http://www.merck4biosciences.com/TBST">www.merck4biosciences.com/TBST</a></td>
</tr>
<tr>
<td>SignalBoost&trade; Immunoreaction Enhancer Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>407207</td>
<td><a href="http://www.emdbiosciences.com/signalboost">www.emdbiosciences.com/signalboost</a> <a href="http://www.merck4biosciences.com/signalboost">www.merck4biosciences.com/signalboost</a></td>
</tr>
<tr>
<td>RapidStep&trade; ECL Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>345818</td>
<td><a href="http://www.emdbiosciences.com/rapidstep">www.emdbiosciences.com/rapidstep</a> <a href="http://www.merck4biosciences.com/rapidstep">www.merck4biosciences.com/rapidstep</a></td>
</tr>		</tbody>
		</table>
		</div>

western blotting: sample preparation to detection

ウエスタンブロット法は、組織ホモジネートまたは抽出物の特定のサンプル中の特定のタンパク質を検出するために使用される分析手法です。それは、ポリペプチドの長さ（変性条件）またはタンパク質の3次元構造（ネイティブ/非変性条件）で、ネイティブまたは変性タンパク質を分離するゲル電気泳動を使用しています。タンパク質を標的タンパク質に特異的な抗体を使用してそれらがプローブされる膜（通常はニトロセルロースまたはPVDF）、（検出）に転送されます。

Watch this Scientific Journal Video about Western Blotting: Sample Preparation to Detection at JoVE.com

Western Blotting: Sample Preparation to Detection

ウエスタンブロット法は、組織ホモジネートまたは抽出物の特定のサンプル中の特定のタンパク質を検出するために使用される分析手法です。

ウェスタンブロッティング：サンプル調製を検出する

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

western-blotting-sample-preparation-to-detection

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JoVE Journal

Biology

138.9K ビュー.  EMD Chemicals Inc.. ウエスタンブロット法は、組織ホモジネートまたは抽出物の特定のサンプル中の特定のタンパク質を検出するために使用される分析手法です。

ビデオ: Western Blotting: Sample Preparation to Detection

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDIサンプル調製：超薄層法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

生物学

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

西洋は、Invitrogen NuPage NOVEXビストリスミニゲルを用いたウェスタンブロット

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

ホルマリン固定からプロテオミクスサンプル調製およびパラフィン包埋組織

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

ウェスタンブロッティングにおける実用的で便利で信頼性の高い全タンパク質ローディング制御のためのV3染色不要のワークフロー

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

町の西部最速：クラシックウェスタンブロット分析に現代風にアレンジ

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

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 ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

質量サイトメトリー分析のための試料調製

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

サンプル準備および透過電子顕微鏡によるエクソソームのイメージ投射

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透過電子顕微鏡用小型サンプル準備

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...