博士ジョーネアン、マットBryman、マイケルボルドー、Ujwal Bagal、Huizheジン、そしてアマンダBouffier：我々は、そのヘルプマツ組織の収集は不可能だったでしょうせずに次の人に感謝の意を表します。

パート1：ツリーの収穫 ツリーを選択し、伐採された後、それは慎重にし、可能な限り迅速に作業することが重要です。それぞれ別の組織の種類が収穫されているサンプルの材料の任意のクロスコンタミネーションを避けるために、彼らは自らの液体窒素容器にすぐに配置する必要があります。このRNAの分離のプロトコルがスケーラブルです。 <ol><li>ボルトの長手方向軸線に続く二、三箇所の樹皮は約0.5〜0.75メートルの長さにし、チェーンソーのスコアとボルトをカット。 </li><li>樹皮が木材から分離し、それが茎から完全に分離するまで樹皮のセクションに戻って引きながらノミを作業を続行し始めるまでの木材のみを使用して、得点領域の端を働く。 </li><li>二次木部は、樹皮から削除されているボルトの長手方向軸に沿ってこすることによりストレートエッジ、片面カミソリで収穫されます。組織の汚染を最小限に抑えるためにゴム手袋を着用してください。 </li><li>樹皮のセクションの内側に配置されている師部は、容易に剥離手動で収穫される。 </li><li>反応の木は、尊敬グラウンドで元の方向を示すために、スプレーペンキでそれらをマークした後、手足の下面から収集されます。 </li><li>手足は1〜2 mのボルトに切断し、反応の木の2つのタイプのおおよその位置に基づいて縦軸に沿って反対側に得点されています：反対の木（四肢の上部側）と圧縮あて材（底上肢の側）。樹皮は、反応の木材の各タイプをカバーする樹皮を一度に削除されることを除いて、上記のような手足から分離されている。二次木部や篩部は、前述のように回収し、液体窒素中に配置されます。 </li><li>このような針、茎頂や若い円錐のような他のサンプルは、、手で収穫するか、または必要に応じ剪定ばさみを使用して設定できます。 </li><li>液体窒素で凍結採取した試料は、顕著な袋または適切な容器に入れ、実験室に戻って出荷のためにドライアイスで梱包してください。 </li></ol>パート2：サンプルのフリーザーミル加工<ol><li>液体窒素でスペックス6850フリーザーミルタンクを記入し、研削バイアルのいずれかにインパクトバーを配置し、プレチルに液体窒素で満たす。液体窒素を捨て、バイアルにサンプルを追加。 </li><li>余分な液体窒素が製粉チャンバー内に残っていないとエンドキャップは、チャンバーを密封するために装着される前に、その過剰な窒素ガスが完全に排気されていることを確認します。 </li><li>キャリアと密接に研削バイアルを挿入します。 </li><li> 14の速度の設定で1分/サイクルの2サイクル用ミル木質サンプル。 </li><li>あらかじめ冷却され、液体窒素を少量含まれているビーカーに粉砕されたサンプル（木粉）を除去するために液体窒素冷却したスパチュラを使用してください。 </li><li>必要になるまで-80℃で貯蔵容器と所定の位置に液体窒素/サンプルスラリーを注ぐ。 </li></ol>パート3：RNA単離、1日目<ol><li> 50mLのキャップファルコンチューブにRNA分離バッファの20mlの代わりとし、400μLのβ-メルカプトエタノール、軽くボルテックスして、60℃の水浴中で熱を加える。 </li><li>各チューブに凍結組織の4gを追加します。フタをし、10秒間ボルテックスし、次いで手で強く振とう。 </li><li>チューブに等量のクロロホルムを追加し、穏やかに転倒混和。回転するホイール​​にチューブを置き、5分間転倒ミキシングが可能。 </li><li> 12,000（17200 × g）で5分間SS34固定アングルローターの回転数で50 mLのオークリッジチューブと遠心分離機に混合物をデカント。 </li><li>新鮮なオークリッジ管に水相を移し、クロロホルムの半分のボリュームを追加します。 </li><li> 1分間ボルテックスし、5分間回転するホイール​​に混和して。 5分間SS34固定アングルローターで12,000 rpmで（17200 × g）で遠心する。 </li><li>新鮮なオークリッジ管に水相を移し、クロロホルムの半分のボリュームを追加します。 1分間ボルテックス。 10分間、SS34固定アングルローターで12,000 rpmで（17200 × g）で遠心する。 </li><li> 50mLの高速ポリプロピレンチューブに水相を移す。 20分SS34固定アングルローターで10,000 RPM（11900 × g）で遠心する。 </li><li>デカントは50mlのファルコンチューブに上清と清の四分の一のボリュームを追加する10 M LiClをソリューション。 4で簡潔にボルテックスし、場所で一晩沈殿のためのC。 </li></ol>パート4：RNAの単離、2日目<ol><li>開始する前に65℃の水浴中で熱のSSTEバッファ。 </li><li> 4℃で30分間SS34固定アングルローターで11000回転（14450 × g）で遠心分離によってポリプロピレンのチューブ及びペレットRNAにLiClを沈殿させ、サンプルを注ぐ℃に</li><li>遠心分離後、オフ注ぐと液体を捨てる、と約1分間、キムワイプで試験管を反転。残りの上清をピペットで。 </li><li>徹底的にサンプルを混合するピペットを用いて加熱SSTEの800μLで各ペレットを再懸濁する。 2 mLのAmbion社のRNase - freeマイクロチューブに再懸濁したサンプルを移す。 </li><li>熱サンプルを65℃の完全な再懸濁を確保するために積極的なボルテックスに続いて2分間。 </li><li>各サンプルチューブに、フェノール - クロロホルム、PH8の等量を追加。 30秒間ボルテックス各サンプルを、その後3分間14,000 rpmで遠心でスピン</li><li>新しい2mLのはAmbion社のRNase - freeマイクロチューブに水相を移し、等量のクロロホルムを加える。 3分間14,000 rpmで遠心で30秒とスピン間ボルテックス各サンプルを。 </li><li>以前に2分間11,000 rpmでmicrofugingによって準備されているフェーズロックジェルチューブに水相を移す。クロロホルムの半分量を加え、穏やかに混合してピペット。 </li><li> 5分間、11,000 rpmで遠心分離し、新鮮な2mLのアンビオンRNaseフリーのマイクロチューブに水相を移す。 </li></ol>パート5：RNAの降水量とエタノールで洗浄<ol><li>水相に50μLの3.0 M酢酸ナトリウム、pH 4.8、1 mLの100％エタノールを加えます。 -80の渦と沈殿-20℃で30分間または一晩℃です。 </li><li>慎重にマイクロ4で30分間14,000 rpmでサンプル° C、そして上清を吸引。ペレットを乱さないでください。 </li><li> 1mLを-20 ° C 70％（v / v）エタノールでRNAペレットを洗浄してください。 1分間14,000 rpmで微量とエタノールを吸引除去する。 </li><li>ステップ5.3を繰り返します。 </li><li> 3-5分のためのベンチでチューブと空気乾燥ペレットを暴露する。 </li></ol>パート6：最終的な再懸濁および定量<ol><li> 100μLのDEPC処理水に各ペレットを再懸濁する。ボルテックスで混合し、氷上に置く前に2分間、65℃でチューブをインキュベートする。サンプルの再懸濁を確保するために必要に応じて繰り返します。 </li><li>希望と分光学的定量のために、10mMのトリス、pH 7.5の10倍希釈液50μLを加えた場合のサンプルのようなプール。 280分の260と230分の260の吸光度比はそれぞれ、一般的に2.1と2.2よりも大きいです。 60120μg/ gの凍結組織からのRNA単離収率の範囲。 </li><li> TAEバッファー中で1％アガロースゲル上で1.5〜2.5μgのRNAを実行してサンプルを分析する。より重要なサンプルについては、製造元の指示に従ってAgilent 2100バイオアナライザを用いてRNAを分析する。 </li></ol>代表的な結果： 木質試料で60〜120μg/ gの凍結組織とは異なる針葉樹の組織の範囲からのRNA単離収率は通常、70〜80グラム/ gの凍結組織の周りに降伏。 280分の260と230分の260の診断率は両方とも2.1よりも一般的に大きくなって、そしてRNAサンプルはそれぞれ、重要なフェノールまたは炭水化物の汚染がないことを良い指標です。反応ステップの染色をアガロースゲル電気泳動によって分析したサンプルは、25S、18S、および5S RNA（図1）の3つの明瞭なバンドを表示する必要があります。アガロースゲル上で18Sに28Sの化学量論を決定することは幾分主観的であり、そしてそのような実行中のゲルのための条件、染色、およびサンプルの負荷などの要因がすべて影響を及ぼす可能性があります。一般的には、28S：18S比が&gt; 1.5であることが表示されます。ただし、一部の針葉樹のサンプルは低い比率を持っている。例えば、Agilent 2100バイオアナライザ分析の後我々は、化学量論は1.2:1に近い方の異なる組織タイプから針葉樹のサンプルの複数のインスタンスを見てきました。このように、見かけの低い28S：18Sアガロースゲル電気泳動による化学量論は、必ずしも低品質のサンプルを示しているわけではありません。葉、茎、およびコーンのサンプルは、しばしば追加の明瞭なバンドが葉緑体リボソームRNAによる発表があります。サンプルウェルまたは高分子量のバンド（&gt; 8〜10キロバイト）から移行しない流します染色材料は、通常、ゲノムDNAのコンタミネーション（図2）を示している。 5Sバンドのまたは下近傍の減少リボソームバンドの染色または明白18Sの低分子量の汚れが&gt; 28Sの化学量論は、RNAの分解が（図3）が発生したことを示す良い指標です。 1.2から2.0にどこまで18S比：Agilent 2100バイオアナライザ電気泳動分析では、ベースラインが低く、18Sと28Sをもつ28SリボソームRNAに対応する主要なピークを持つ比較的滑らかでなければなりません。 AgilentのRNAの完全性番号（RIN）値は、RNAの品質の良い予測因子である可能性があり、マイクロアレイのターゲットの調製に使用するRNAの少なくとも7以上にする必要があります。 18S：図5は、28Sで明らかにしたように非常に高いベースラインと顕著な低下を持っている貧しい木部RNAプレップを示し、18S 1.4〜等しい比と8.6のRIN値：図4は、28Sと木部のRNAの典型的なAgilent 2100バイオ電気泳動図を示しています。 RA0.65〜3.4のRINの値に等しいチタン。 <img src="/files/ftp_upload/1751/1751fig1.jpg" alt="図1" /> 高品質な松のRNAの図1。アガロースゲル分析。レーン1、NEB 1キロバイトの標準は、レーン2は、特徴的なリボソーム28S、18S、そして5Sのバンドと明らかに28Sとテーダマツ二次木部からの典型的なトータルRNAの分離を示しています：18S比&gt; 1。 <img src="/files/ftp_upload/1751/1751fig2.jpg" alt="図2" /> ゲノムDNAで汚染された松のRNAサンプルの図2。アガロースゲル分析。レーン1、NEB 1キロバイト標準、レーン2、ゲノムDNAのコンタミネーションを示す木部RNAサンプル。 <img src="/files/ftp_upload/1751/1751fig3.jpg" alt="図3" /> ゲノムDNAとRNAの分解やコンタミネーションを示す松サンプルの図3。アガロースゲル分析。レーン1、NEB 1キロバイト標準、Lane2、ゲノムDNAのコンタミネーションと深刻なRNAの分解を示す木部RNAサンプル。 <img src="/files/ftp_upload/1751/1751fig4.jpg" alt="図4" /> 図4。木部のトータルRNAのAgilent 2100バイオアナライザ電気泳動図は、テーダマツの木部に記載された方法を用いて単離した。 28S：18S比は、RIN値は8.6に等しく、1.4に等しい<img src="/files/ftp_upload/1751/1751fig5.jpg" alt="図5" /> 図5。深刻な劣化とゲノムコンタミネーションを示す心尖先端トータルRNAのAgilent 2100バイオ電気泳動図。 28S：18S比はRINの値が3.4と等しい、0.65に等しい

<ol>
	<li>Chang, S., Puryear, J., Cairney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

針葉樹の種から高品質のRNAを得ることは木質の組織で検出されたフェノール及び多糖類化合物を高レベルで与えられる困難な作業になる場合があります。 Changらによって開発されたプロトコルを使用して起動。 （1）、我々はより厳密な抽出を発見し、様々な木質と針葉樹種の多種多様なサンプリング非木質組織から非常に高品質のトータルRNAの一貫性のある分離する可能性の手順をクリー?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
</tr>
<tr>
<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
</tr>
<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

このようなテーダマツ（のような特に針葉樹の種、マツ科のファミリーに属するものから分離された組織、<em&gt;テーダマツ</em&gt; L.）、RNAの精製を妨害するフェノール化合物と多糖類の高濃度が含まれています。これらの種から高品質のRNAの単離は、マイクロアレイや他のゲノム解析のために十分な品質のRNAを分離するために、複数の有機溶媒抽出のステップから構成されるRNAの分離のプロトコルのフィールドと雇用の厳格な組織の収集手順が必要です。フィールドで収集テーダマツのサンプルから高品質のRNAの分離は非常に結果を改善に挑戦、しかしいくつかの標準的な組織の変更やRNAの分離の手続きができます。サンプルが正常に特に大規模な収穫時に、フィールドから収集し、輸送されていない場合、一般的なRNA分解の程度が増加する。トータルRNAの収量はサンプルがwoodyの組織から来た場合は特に、分離をRNAに前に液体窒素の冷凍工場でサンプルを粉砕して大幅に増加することができます。これは主にこのようなフェノール化合物、および両方のほとんどの針葉樹種の木質組織からの抽出物中の高濃度で存在する多糖類などの酸化剤の存在、によるものです。削除しない場合、これらの汚染物質は、そのようなRNAの分解、低収量と低品質RNAサンプルでその結果としての問題、につながる以上運ぶことができる。フェノール化合物だけでなく、多糖類のキャリーオーバーは、また減らすか、完全に逆転写酵素または一般的なcDNA合成に使用される他のポリメラーゼの活性をなくすことができます。研究者が期待するなら、特に、c​​DNAライブラリーの世代の二本鎖cDNA合成のテンプレートとして使用される運命RNA、PCRや定量PCRのために、またはマイクロアレイターゲット材料の合成のための一本鎖cDNA合成は、最高品質のものでなければならない最適な結果を得るために。一般的に他の多くの植物種に用いるRNAの分離技術は、針葉樹のサンプルからこれらの汚染物質を除去するため、下流の操作に適したトータルRNAサンプルを得られない能力がしばしば不十分である。このビデオでは、篩部の収穫、二次木部、およびあて材の木部に、40年前の木の伐採をはじめ、針葉樹の組織のフィールドのコレクションのための方法を示しています。我々はまた、一貫して、その後の酵素的操作のための高品質のRNAをもたらしましたRNAの分離のプロトコルを示しています。

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

テーダマツから篩部および木部を含む多くの植物組織、（<em&gt;テーダマツ</em&gt; L.）、フェノール類およびRNA精製を妨害する多糖類の高レベルが含まれています。このプレゼンテーションでは、屋外栽培の組織やマイクロアレイや他のゲノム解析のための十分な品質のRNAの単離の収穫のためのテクニックを説明します。

ロボロリーパインからのRNA分離の改良法（ P. taeda L.）及びその他の針葉樹種

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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JoVE Journal

Biology

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

13.7K ビュー.  University of Georgia (UGA). テーダマツから篩部および木部を含む多くの植物組織、（テーダマツ L.）、フェノール類およびRNA精製を妨害する多糖類の高レベルが含まれています。このプレゼンテーションでは、屋外栽培の組織やマイクロアレイや他のゲノム解析のための十分な品質のRNAの単離の収穫のためのテクニックを説明します。

ビデオ: An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

ロボロリーパインPtGen2 cDNAマイクロアレイの処理

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

遺伝子発現解析のための胚ゼブラフィッシュとcDNA合成からのRNA分離

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル- L</subキャプチャの複合質量分析法（CCMS）別> -ホモシステイン結合タンパク質

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

キネシンの単離及び精製からショウジョウバエ胚

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

から細胞外液のヴァンパイア·アイソレーション<em&gt;線虫（Caenorhabditis elegans）</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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 ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

マウス膵臓からのRNAの単離：リボヌクレアーゼが豊富な組織

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

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 ...

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

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 often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

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 ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

マウス初代肝細胞単離のための時間と労力の効率的なプロトコルの改善

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...