ダニエル陳からデジタル ビデオ生産チームのブッフホルツ高校編集ビデオで支援に感謝いたします。この気孔細胞・ プロテオミクス ・ メタボロミクス陳の実験室での研究は、米国国立科学財団 (0818051、1158000、1412547) からの補助金によってサポートされています。Wenwen 香港は中国公費によってサポートされます。博士 Qiuying パンをサポートするには、中国の奨学金委員会と国家自然科学基金、中国の (31570396)。

1. 成長する植物
<ol><li>Potting の混合物の種を発芽させます。植物を育てる成長室 140 µmol 光子 m− 2の− 1の光強度と光速で 8 h 22 ° C で 16 時間日長で暗い、18 ° C で 2 週間。</li>
	<li>4"直径の鍋土を含むに個別に類似サイズの苗を移植し、手順 1.1 で説明した追加同じ条件で 3 週間の成長します。 注: は、湿った土壌を維持する週 2 回水道水で植物を水します。</li>
</ol>2. 豊かな気孔の準備
<ol><li>5 週間の古いシロイヌナズナ植物メスと個々 の葉を削除します。</li>
	<li>裏面 (下側) に付着したワンピースと葉の向軸 (上) 側に付着した他の作品と明確スコッチ テープの 2 つの部分に各葉を取り付けます。5 葉のテープを残す s。</li>
	<li>優しい皮をむく離れて舗装と細胞と葉肉細胞の向軸側背軸側を別々 にそれぞれの手の人差し指と親指を使用してテープの 2 つの部分。</li>
	<li>気孔を開くの 60 mL に葉の背軸側から皮バッファー (50 mM KCl、10 mM MES 島、pH 6.2 1 M コに調整) 40 × 12 mm サイズ ペトリネットの場所は、収集された料理します。<ol>
<li>すべてのサンプルの皮が収集されるまでは、上記の手順を繰り返します。</li>
	</ol>
</li>
	<li>手順 1.1 で述べた光の条件下で生育室に皮シャーレを配置します。気孔が全開に 2 h の光の下で皮を残します。</li>
	<li>場所は、150 mm × 20 mm にペトリ皿細胞壁の消化酵素の混合 (0.7% セルラーゼ R-10、0.025% macerozyme R-10、0.1% (w/v) ポリビニルピロリドン-40、および 55% 基本的なソリューション (0.55 M ソルビトール、0.5 mM で 0.25% (w/v) ウシ血清アルブミン 50 mL を含む皮します。CaCl2, 0.5 mM MgCl2、0.5 mM アスコルビン酸, 10 μ M KH2PO4, 5 mM 4-morpholineethanesulfonic 酸 (MES) ph 5.7 1 M コで調整)。</li>
	<li>20 分間 50 rpm でシャーレに相互シェーカーで皮を振る。</li>
	<li>20 分は、50 mL の水を含む 150 × 20 mm サイズのシャーレに皮を転送した後、皮をピンセットで酵素液から削除します。</li>
	<li>15 の皮を洗う転送ピペットを使用して、任意の残留酵素液を削除する水 10 mL で 2 回 s。</li>
	<li>手順 2.4 で使用されるバッファーを開いて新鮮な気孔の 50 mL の 150 mm × 20 mm サイズのシャーレに皮を配置するのにピンセットを使用します。手順を回復する気孔を許可する 1.1 で述べた光の条件で 1 時間インキュベートします。</li>
</ol>3. アブシジン酸 (ABA) 処理と気孔運動アッセイ
<ol><li>で 150 mm × 20 mm サイズ シャーレ。15 分 50 mL の気孔開口バッファーまたは適切な治療時間の 10 μ M の ABA の最終濃度と気孔開口バッファーの 50 mL 30 ° C で豊かな気孔皮を孵化させなさい。</li>
	<li>5, 15, 30 分間で 50 rpm シェーカーに皮を配置します。各時間間隔後ピンセットで皮を削除、手順 3.3 で画像処理の手順に従います。</li>
	<li>ABA 処理後光学顕微鏡で気孔 ABA 処理前に、と様々 な時点での画像を取る。<ol>
<li>皮を取るし、ガラス顕微鏡スライド上に置きます。</li>
		<li>顕微鏡ステージにスライドを配置し、皮のイメージを視覚化できるので、ステージを調整します。</li>
		<li>罰金を調整し、ガード細胞の鮮明な画像を得るために、顕微鏡の焦点をコースします。</li>
		<li>40 倍の倍率で複数の気孔のいくつかの画像を取る。</li>
	</ol>
</li>
	<li>ImageJ8を使用して 60 の気孔開口部を測定します。</li>
	<li>標準のエラーおよび p 値 < 0.05 60 の気孔開度測定の意義を計算します。</li>
</ol>4. 蛋白質の抽出および SDS ページの分離
<ol><li>15 の皮を挽くチルド乳鉢と乳棒を使用して皮をカバーする十分な液体窒素で s。</li>
	<li>50 皮ごと Tris 飽和フェノール (pH 8.8) の 3 mL を加えると 3 mL タンパク質抽出バッファー (0.9 M ショ糖 0.1 M トリス-HCl、0.01 M EDTA, 0.4 %2-メルカプトエタノール、プロテアーゼ、ホスファターゼ阻害剤は 1 mM の 10 μ L)、モルタルにピペットで 5 エディーの皮を挽くとヒューム フードに至り分。</li>
	<li>ピペットを使用して抽出を転送し、皮、オークリッジに金属製のピンセットを使用してチューブを遠心し、4 ° C で 1 時間を 50 rpm でシェーカーの扇動</li>
	<li>オークリッジ遠心管から皮を外し、5,000 × g で 10 分間で抽出物を遠心分離します。ピペットを使用して新しいきれいなマイクロ遠心チューブ トップのフェノール相を転送します。</li>
	<li>フェノールの沈殿物は 100% メタノールで冷たい 0.1 M 酢酸アンモニウム 5 ボリュームを追加することによってタンパク質を抽出しました。渦を簡潔にし、-20 ° C で一晩インキュベート</li>
	<li>4 ° C、20 分で 20,000 × g で遠心分離機、ゆっくりとチューブからそれを注ぐことによって、上清をデカントし、管内の蛋白ペレットを保持します。</li>
	<li>メタノール、酢酸アンモニウム 0.1 M で二回蛋白質餌を洗浄し、80% アセトンで 2 回と 100% 冷アセトン。 注: 洗浄は、タンパク質のチューブに指定された試薬 10 mL を追加することによって行われます。5 分、10 分の 4 ° C で 15,000 × g で遠心分離後 50 回転でシェーカーを揺り動かしなさい。その後、蛋白ペレットのみを保持チューブから試薬を注ぐ。</li>
	<li>ペレットに 100% 冷アセトン 1 mL を追加し、上下にゆっくりとピペッティングによって再サスペンドします。</li>
	<li>タンパク質サスペンション ピペット 2 mL マイクロ遠心チューブに転送し、5 分のための 4 ° C で 20,000 × g で遠心分離機します。</li>
	<li>チューブからデカントでアセトンを削除し、10 分の発煙のフードでペレットを乾燥します。</li>
	<li>溶解バッファー (8 M 尿素、0.5 %sds、ph 8.5 30 mM トリス-HCl) の 200 μ L マイクロ遠心チューブにタンパク質を溶解とで 30 分間遠心 15 ° C 20 分、収集新しい管の上澄みで 20,000 × g、渦。</li>
	<li>次のタンパク質定量プロトコル9タンパク質濃度を定量化します。</li>
	<li>プロトコル10次ゲル電気泳動蛋白質の分離のための蛋白質のサンプルを使用します。</li>
</ol>

<ol>
	<li>Acharya, B., Assmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+interactions+in+stomatal+function.">Hormone interactions in stomatal function.</a> Plant Molecular Biology. 69 (4), 451-462 (2009).</li><li>Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., Waner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+cell+signal+transduction.">Guard cell signal transduction.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 52, 627-658 (2001).</li><li>Obulareddy, N., Panchal, S., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+Cell+Purification+and+RNA+Isolation+Suitable+for+High-Throughput+Transcriptional+Analysis+of+Cell-Type+Responses+to+Biotic+Stresses.">Guard Cell Purification and RNA Isolation Suitable for High-Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses.</a> Molecular Plant-Microbe Interactions. 26 (8), 844-849 (2013).</li><li>Pandey, S., Wang, X. Q., Coursol, S. A., Assmann, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+applications+of+Arabidopsis+thaliana+guard+cell+protoplasts.">Preparation and applications of Arabidopsis thaliana guard cell protoplasts.</a> New Phytologist. 153 (3), 517-526 (2002).</li><li>Schroeder, J. I., Raschke, K., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+Dependence+Of+K++Channels+In+Guard-Cell+Protoplasts.">Voltage Dependence Of K+ Channels In Guard-Cell Protoplasts.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (12), 4108-4112 (1987).</li><li>Wu, F. H., 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=Tape-Arabidopsis+Sandwich+-+a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method.</a> Plant Methods. 5, 10 (2009).</li><li>Svozil, J., Gruissem, W., Baerenfaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+targeting+of+proteins+in+Arabidopsis+leaf+mesophyll,+epidermal+and+vascular+tissues.">Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.</a> Frontiers in Plant Science. 6, (2015).</li><li>Schneider, C., Rasband, W., Eliceiri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Agnew, B., Murray, D., Patton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+solid-phase+fluorescence-based+protein+assay+for+quantitation+of+protein+electrophoresis+samples+containing+detergents,+chaotropes,+dyes,+and+reducing+agents.">A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents.</a> Electrophoresis. 25 (15), 2478-2485 (2004).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> Bio-protocol. Bio101 (80), (2011).</li><li>Thompson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+mass+tags:+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS+(vol+75,+pg+1895).">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS (vol 75, pg 1895).</a> Analytical Chemistry. 78 (12), 4235-4235 (2006).</li><li>Wiese, S., Reidegeld, K. A., Meyer, H. E., Warscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+labeling+by+iTRAQ:+A+new+tool+for+quantitative+mass+spectrometry+in+proteome+research.">Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research.</a> Proteomics. 7 (3), 340-350 (2007).</li></ol>

利害の対立が宣言されていません。

ガード細胞は植物におけるシグナル伝達機構を研究するためのモデル システムを研究で使用される試料の調製が生物的質問に答えるに最も適切なことを確認することが重要です。植物研究コミュニティの中で細胞の増加の関心にもかかわらずは普遍的な方法が気孔運動と生理の両方できるようになる細胞を準備する方法の 1 つで検討する分子レベルの変化だけでなく、システム。それらを分...

気孔は、全体の生理学と植物のフィットネスに重要な役割を果たします。これらの小さな植物の特定の構造、細胞として知られている 2 つの特殊な表皮細胞によって形成され、葉の切断面に最も豊富にあります。気孔は、蒸散による水損失の制御と同様、植物と大気間のガス交換の必要があります。細胞調節する異なる外部 (例えば光、湿度、病原体の接触、二酸化炭素のレベル) の統合により気孔開口部および内部 (e.g。、内因性ホルモン) 刺激1,2。過去 20 年間で、この型のセルを勉強セルシグナ リング プロセス植物のためのモデル システムとなりました。これらの細胞は葉; 総細胞のごく一部を構成します。したがって、彼らのユニークな携帯を調べるためには、生化学的および分子プロパティ単一セルにそれ、他の葉の細胞型からそれらを分離する必要。過去には、そのガード細胞研究はしばしば細胞プロトプ ラスト (GCP)3,4、5の準備を関与しています。これは通常大量のブレンドによる機械的破壊や、細胞壁分解酵素の使用を必要とし、少し収量を倍のコストと時間のかかる一般的に GCP サンプルを頻繁に準備に多くの時間がかかるように証明されています。もっと重大に、細胞は、気孔開口を調節する完全なペアとして機能、GCP の使用は細胞シグナリングを勉強するための人工的なシステムとして見ることができます。
ここでは、気孔そのまま細胞が濃縮されているし、刺激に対する生理反応を維持を準備する方法を開発しました。このメソッドは、葉肉細胞プロトプ ラスト6,7を分離するために使用された方法に触発されました。本手法は, 気孔は、明確な舗装と細胞を含む裏面層から葉の向軸レイヤーに接続されている最初葉肉細胞の大半を分離するスコッチ テープを使用して用意しています。これは葉の両側にテープを貼付し、それらを離れて剥離です。裏面層から細胞が気孔開口部バッファー内の光の下で回復が許可されます。後残りの舗装の細胞が細胞壁分解酵素、テープに濃縮されているガード細胞を残しての少量を使用して削除されます。
このシンプルなメソッドで応答性の高い有効な気孔のガード細胞迅速かつ効率的に、最小限のコストで 50 の皮から濃縮細胞を取得するより少ない時間を取って準備します。ここにメソッドは、植物細胞のプロトプ ラストが用意されて、従来よりも以下のダメージを課しています。さらに、材料はこのメソッド収量の望ましい蛋白質量から収集されます。最も重要なは、葉がブレンドにさらされていないか、長い消化時間と細胞のまま、ための研究結果より密接にガード セルの自然な生物学に関連します。この方法では、気孔運動関連付けることができますリアルタイムで分子レベルで変更します。したがって、この製法を用いた研究から得られた知識は、ガード細胞シグナル伝達のより包括的な理解のため重要となります。
モデルとして植物のシロイヌナズナを用いてください。ただしこのプロトコルは消化時間の若干の修正をナタネなど他の植物の気孔のガード細胞の濃縮に適用できます。概念の証拠として、このメソッドを介して濃縮細胞は、実行可能なフルオレセイン ・ ジアセテート (FDA) 試金および植物ホルモンのアブシジン酸 (ABA) をそれぞれ活用研究に示すように刺激に反応しました。さらに、高品質のタンパク質がこれらのガードの細胞から分離することができますを確認しました。ここでは、このプロセスの詳細なプロトコルについて述べる。

<table border="0" cellpadding="0" cellspacing="0" style="width:889px;" width="889">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Stainless Steel Surgical Scalpel</td>
			<td style="width:297px;">Feather&#160;</td>
			<td style="width:119px;">&#160;NC9999403</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Scotch Tape (3M)</td>
			<td style="width:297px;">ULINE</td>
			<td style="width:119px;">S-9781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Petri Dish</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td>BR455701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase (Onozuka R-10)&#160;</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macerozyme R-10</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DM6000B Microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Protease and Phosphatase Inhibitor Cocktails</td>
			<td style="width:297px;">Thermo Fisher Scientific&#160;</td>
			<td>&#160;PI78443</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">Oak Ridge High-Speed PPCO Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3119-0030</td>
			<td></td>
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			<td height="21" style="height:21px;">EZQ Protein Quantitation Kit</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>R33200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescein Diacetate (FDA)</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>F1303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Abscisic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4906</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Metro Mix 500</td>
			<td style="width:297px;">BWI Companies&#160;</td>
			<td style="width:119px;">TX-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Laemmli Sample Buffer</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0737</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Bio-Safe Comassie (G-250)</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0786</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Microcentrifuge Tube (2mL)</td>
			<td style="width:297px;">USA Scientific, Inc.</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">ImageJ software</td>
			<td style="width:297px;">National Institute of Health</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Tweezers&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4017-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">12% Mini-PROTEAN TGX Precast Gel</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">456-1043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope slides</td>
			<td style="width:297px;">Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
	</tbody>
</table>

stomata tape-peel: an improved method for guard cell sample preparation

細胞の研究は、植物の全体的な機能にこのセルの型の特定の貢献の知識に不可欠です。多くの場合分離することは困難だし、挑戦を提供する従ってそれらをもっと勉強します。
本研究では、細胞濃縮法を確立します。プロトコルは、スコッチ ガードのセルを隔離し、セルからの蛋白質の抽出を採用しています。このメソッドは、分離中に整合性と細胞の収量を向上し、細胞シグナリング プロセスと気孔の表現型の関連性の研究に信頼性の高いサンプルを提供します。この方法では植物の葉はテープの 2 つの部分間で分割され背軸側を削除するには、離れて皮をむいた。このプロトコルは、シロイヌナズナ葉の組織細胞を分離するために適用されました。セルは、フルオレセイン ・ ジアセテートの生存率を決定する (FDA) とアブシジン酸 (ABA) ABA に気孔運動を評価するために扱われました。結論としては、このプロトコルは蛋白質の分離と濃縮細胞を準備するため有用証明します。有効にする生理学的およびその他の生物学的研究とタンパク質・細胞の品質はここで得られました。

シロイヌナズナの代表的なイメージは前に、と後、葉肉細胞や表皮細胞の消化は、図 1に示すセルをガードします。ガード セル実行可能性前に、と葉肉細胞と表皮細胞の除去後は、酵素活性と細胞膜の完全性 (図 1 bと1 D) を測定する FDA を使用して観察できます。図 2は、ABA の処置への応?...

Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

このプロトコルでは、生理学的およびその他の生物学的研究に役立つ濃縮細胞を準備する方法について説明します。

気孔テープ剥離: 細胞試料の調製法の改良

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

stomata-tape-peel-an-improved-method-for-guard-cell-sample-preparation

Research

JoVE Journal

Biology

14.9K ビュー.  University of Florida. このプロトコルでは、生理学的およびその他の生物学的研究に役立つ濃縮細胞を準備する方法について説明します。

ビデオ: Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

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

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

アセンブリ、読み込み、および分析用超遠心器のサンプルセルの整列

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

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

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 ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

品質イノシトールピロリン酸塩の調製

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

飛行性能の正確かつ迅速な測定のための改善された方法での<em&gt;ショウジョウバエ</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

単一のビリオン原子間力顕微鏡と超解像蛍光イメージングのサンプル調製

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

免疫組織化学および免疫蛍光分析のための腸組織の調製のための改良されたスイス・ローリングテクニック

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

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 ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM分析のための低細胞数とサンプル調製のための代替的アプローチ

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

ハイブリッドカット：手に負えない植物組織試料のための改善されたセクショニング方法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...