This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

マウスを用いた実験手順は、マギル大学（プロトコル4966）の動物実験委員会によって承認された。 培養細胞からのタンパク質抽出物の調製<ol><li>氷冷したリン酸緩衝生理食塩水（PBS）10mlで二回培養された細胞を洗浄する。 </li><li>ラバーポリスまたは1.5 mlマイクロ遠心チューブに氷冷PBS、転送懸濁液1ml中にプラスチック製のセルスクレイパーのいずれかで付着細胞をこすり。 </li><li> 4℃で700×gで5分間のマイクロ遠心でスピン、。吸引するPBS。 </li><li> 10 7個の細胞あたりの氷冷細胞質溶解バッファー（ 表1）の100μlのを追加し、上下にピペット。 </li><li> 20分間氷上でインキュベートする。 </li><li> 4℃での微量遠心で最高速度で10分間スピン。 </li><li>ペレットを捨てる。新しい1.5 mlマイクロチューブに上清を移し、氷上に保つ。 </li><li> DETブラッドフォードアッセイ23を使用して- （を10μg/μL通常1）アーミンタンパク質濃度。 </li><li>使用するまで-80℃でアリコートと店舗細胞抽出物。 </li></ol>マウス肝臓および脾臓からのタンパク質抽出物の調製<ol><li> CO 2吸入でマウスを安楽死させる。 </li><li>解剖ボード上のきれいなパッドの上に安楽死させた動物を置きます。はさみで腹部を開きます。 </li><li>ハサミとピンセットを使用して、肝臓および脾臓を分析し、およそ50 mlの氷冷PBSで各組織をすすぐ。 </li><li>すぐにメスで小片に組織をカットし（例：約1〜2ミリメートル3）。 </li><li>遅滞なく、新鮮なクライオチューブ内の組織の作品を配置した後、液体窒素でそれらを凍結スナップ。ストアスナップ凍結組織のアリコートを-80℃で使用するまで。 </li><li>の0.25〜0.5ミリリットルに- （2〜3約1）凍結組織の一枚を均質化する10秒間、組織ホモジナイザーを氷冷細胞溶解緩衝液（ 表1）。 </li><li> 20分間氷上で1.5ミリリットルマイクロ遠心チューブと寒さにホモジネートを転送します。 </li><li> 4℃での微量遠心で最高速度で10分間スピン。 </li><li>ペレットを捨て、新しい1.5ミリリットルのマイクロ遠心チューブに上清を移す。氷の上に保管してください。 </li><li>ブラッドフォードアッセイ23を使用して- （を10μg/μL通常1）タンパク質濃度を決定します。 </li><li>使用するまで-80℃でアリコートと店舗細胞抽出物。 </li></ol>放射標識IRE-プローブの調製<ol><li>制限エンドヌクレアーゼXbaIで（プラスミドのμg当たり1 U）、IRE配列の下流を切断すると、1時間37℃でインキュベートすることにより、IRE-含むプラスミドI-12.CAT 22を線形化する。線状化したプラスミドをインビトロ転写の鋳型として使用される。 </li><li>セットアップnは全量20μlのin vitro転写反応において 。 表2に示したストック溶液を使用して追加します。1μlの線形化されたプラスミド鋳型、4μlの転写緩衝液; ATP / CTP / GTPミックス1μlのミックス、10μlの[α-32 P] -UTP、2μlのジチオスレイトール、1μlのRNase阻害剤と1μlのT7 RNAポリメラーゼ。ピペッティングにより混和する。 </li><li> 1時間24 40℃でインキュベートする。 </li></ol>放射標識IRE-プローブ4.精製<ol><li>ピペッティングにより、0.5M EDTA、pH8のミックスを1μlを追加することによって、 インビトロ転写反応を終了します。 </li><li>より良い沈殿のためのキャリアとして、10 mg / mlのtRNAの10を添加する。ピペッティングにより混和する。 </li><li> 3 M酢酸アンモニウムの82.5を添加する。ボルテックスで混ぜる。 </li><li>エタノールの273を添加する。ボルテックスで混ぜる。 </li><li>室温で5分間立ってみましょう。 </li><li> RTで微量全速で10分間スピン。上清を捨てる。 </li><li>ウォッシュは、70％エタノール100μlのペレット。 </li><li> RTで微量全速で10分間スピン。上清を捨てる。 </li><li> 10分間空気乾燥ペレット。 </li><li>二回蒸留、以前にオートクレーブし、H 2 O100μlの再懸濁ペレット</li><li>使用するまで-80℃で液体シンチレーションカウンターで放射能、アリコートの放射性標識IREプローブおよびストアを定量化する。凍結アリコートを、3週間まで使用することができる。 </li></ol> EMSAのネイティブポリアクリルアミドゲルの5準備<ol><li> 1.5ミリメートルスペーサーと櫛を使用してゲル（16×16 cm）を組み立てます。 </li><li> 表3に示したストック溶液を使用し、6％未変性ポリアクリルアミドゲルを調製するために、7.5ミリリットルの40％アクリルアミドミックス：ビスアクリルアミド、5×TBE 5mlの37.5ミリリットル二重蒸留H 2 O </li><li> 10％の新たに調製された過硫酸アンモニウム（APS）と25μlのテトラメチルエチレンジアミン（TEMED）の0.5ミリリットルを追加します。 </li><li>すぐにゲルへのアクリルアミド溶液を注ぎ、それが重合してみましょう。約30分間待ちます。 </li><li>ゲル電気泳動装置を組み立て、0.5×TBEでタンクを記入し、電源に接続します。 </li></ol> 6.電気泳動移動度シフトアッセイ<ol><li> 10μlの（より低いタンパク質濃度を用いてもよい）で総体積細胞溶解緩衝液（ 表1）を用いて細胞または組織からのタンパク質抽出物25μgを希釈する。氷の上に保管してください。必要に応じて、11μlの追加：25：4を休眠IRP1（最終濃度2％）を活性化するために2-メルカプトエタノール（2-ME）で希釈した。 </li><li>二重蒸留H 2 O 200,000 CPM /μL、95℃での熱変性に放射性標識されたIREプローブを希釈する1分間のCと、少なくとも5分間、室温で冷却。 </li><li>タンパク質抽出物に放射性標識されたIREプローブの1μLを添加することにより、EMSA反応をセットアップします。 </li><li>室温で20分間インキュベートする。 </li><li> （プローブ9との非特異的タンパク質相互作用を阻害するために）、さらに10分間インキュベーションを継続し、反応を50 mg / mlのヘパリンの1μlを添加する。 -80℃でストックヘパリンの溶液（50mg / ml）とストアを分注し。 </li><li>長い放射性標識プローブ（&gt; 60ヌクレオチド）を使用する場合、1μlのRNase T1（1 U /μl）を追加し、室温で10分間インキュベートしたプローブへの非特異的タンパク質結合を減少させるため、およびRNA /タンパク質の良好な分離を可能にする電気泳動中に複雑。 </li><li>ローディングバッファー（80％グリセロール+ブロモフェノールブルー）の3μl加え、混ぜると、6％非変性ポリアクリルアミドゲル上にロード。 </li><li> 130 V（5 V / cm）ので60分間ゲルを実行します。 </li><li> Transfer大きな濾紙上に乾燥ゲル。 </li><li>フィルムに露出させ、オートラジオグラフィーを開発しています。露光時間は、1時間（あるいはそれ以下）からO / Nまでの範囲であり得る。 </li></ol>

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The authors declare that they have no competing financial interests.

ここで、我々はIRP1及びIRP2のIRE-結合活性を研究するために開発されたプロトコルを記述し、我々は代表的なデータを示している。異なるプローブを使用することによって、このプロトコルは、他のRNA結合タンパク質の研究のために調整することができる。重要なステップは、プローブのサイズです。正確な結合部位が不明な場合に共通している長いプローブの使用方法は、無料のRNAとは異な?...

EMSAは、もともとの標的DNA配列1,2-有するDNA結合タンパク質の結合を研究するために開発された。原理は、この記事の焦点であるRNA /タンパク質相互作用3、についても同様である。簡単に言えば、RNAが負に帯電され、ポリアクリルアミド（またはアガロース）ゲル中で非変性電気泳動中に陽極に向かって移動します。ゲル内の移行は、その電荷に比例する、RNAのサイズに依存します。 RNAへのタンパク質の特異的結合は、その機動性を変化させ、そして複合体は、無料のRNAに比べてゆっくりと移動。これは主に、分子量の増加だけでなく、電荷およびおそらく立体配座の変化によるものである。プローブとして標識されたRNAを利用する「ゲル遅延」や「バンドシフト」の容易な監視を可能にする。 32 P標識RNAプローブの使い方は非常に一般的であり、高感度を提供しています。 RNA /タンパク質複合体および遊離RNAの移行が検出されるオートラジオグラフィーによる。欠点は、放射能の仕事、および潜在的なバイオセーフティの懸念のための放射能のライセンスとインフラの要件、32 P（14.29日）の半減期が短いことによる放射線分解へのプローブの品質の漸進的な劣化である。したがって、RNAプローブを標識するための代替の非同位体の方法は、蛍光または化学発光イメージング4,5による検出を可能にする蛍光団又はビオチン、とのインスタンスのために、開発されてきた。これらの方法の限界は、同位体標識、およびRNA /タンパク質相互作用を妨害するための非同位体標識の可能性と比較してより高いコストと多くの場合減少した感受性である。非変性ポリアクリルアミドゲルは、ほとんどのEMSAの用途に適していると一般的に使用される。機会に、アガロースゲルは、大きな複合体の分析のための代替手段をもたらすことができる。 EMSAの主な利点は、シンプルさ、感度、および堅牢性4を組み合わせたということです&lt;/ SUP&gt;。アッセイは、数時間以内に完了することができ、洗練された機器を必要としません。 RNA /タンパク質相互作用は、0.1 nMもしくはそれ未満という低い濃度でEMSAによって検出され、結合条件（pHは4.0から9.5、一価の塩濃度が1から300 mMの、温度0から60°C）の広い範囲にすることができる。 RNA /タンパク質複合体の形成はまた、フィルター結合アッセイによって研究することができる。フリーRNAプローブ6を通過する間に、これは、ニトロセルロースフィルターでRNA /タンパク質複合体の保持に基づいて、簡単、迅速、かつ安価な手順である。 EMSAと比較して、そのRNAプローブは複数の結合部位が含まれているか、または粗抽出物は、同じ部位でプローブと結合する二つ以上のRNA結合タンパク質を含む場合には制限されている。複数のRNA /タンパク質相互作用は、フィルター結合アッセイによる検出を免れるであろうが、それらは容易にEMSAによって可視化することができる。いくつかのケースでは、可視化は前夜ですn個の可能な場合、ゲル上でさらに位相差を生じ、EMSA反応のRNA結合タンパク質のいずれかに対する抗体を添加することによって共移行（例えば、ヒトIRP1 / IRE及びIRP2 / IRE複合体）は、2つのRNA /タンパク質複合体（ 「スーパーシフト」）7。 EMSAは、広く鉄代謝8-10の転写後調節因子である、IRP1及びIRP2を研究するために使用されてきた。彼らはいくつかのmRNA 11のUTRを内のIRE、系統発生的に保存されたヘアピン構造に結合することによって作動する。 IREは、最初フェリチン12及びトランスフェリン受容体1（TfR1）13、それぞれ鉄貯蔵および取り込み、タンパク質をコードするmRNAで発見された。その後、IRESは赤血球固有のアミノレブリン酸シンターゼ （ALAS2）14、ミトコンドリアのアコニターゼ15、フェロポーチン16、 二価金属トランスポーター1（DMT1）17、低酸素誘導因子2で発見された<eM&gt;α（HIF2α）18、および他のmRNA 19-21。 UTR「TfR1のmRNAはその3で複数のIREが含まれていますが、UTR「プロトタイプH-およびL-フェリチンmRNAは、それらの5で1 IREが含まれている。 IRE / IRPの相互作用は、具体的には、立体的に43Sリボソームサブユニットの関連付けをブロックすることによりフェリチンmRNAの翻訳を阻害。また、それらは、エンドヌクレアーゼ的切断に対してTfR1 mRNAを安定化する。 IRP1及びIRP2を共有広範な配列類似性と鉄飢餓細胞で高IRE-結合活性を示す。 IRP2がプロテアソーム分解を受けながら鉄充実した細胞では、IRP1は、そのIRE結合活性を犠牲にして細胞質ゾルアコニターゼに変換キュバンのFe-Sクラスターを組み立てる。従って、IRE / IRP相互作用は、細胞の鉄の状態に依存するだけでなく、そのようなH 2 O 2、一酸化窒素（NO）または低酸素症のような他の信号によって調節される。ここでは、Eによって粗細胞および組織抽出物からIRE結合活性を評価するためのプロトコルを記述しMSA。我々は、IRE配列が元々下流T7 RNAポリメラーゼ部位のセンス方向に導入されたプラスミドのDNAテンプレート（I-12.CAT）からのインビトロ転写によって生成した32 P標識H-フェリチンIREプローブを使用アニーリングした合成オリゴヌクレオチド22のクローニング。

<table border="0" cellpadding="0" cellspacing="0" width="430">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
			<td style="width:117px;">Catalog Number&#160;</td>
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			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
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			<td>SIGMA</td>
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			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
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		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
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		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
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			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
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		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
	</tbody>
</table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA /タンパク質相互作用は、転写後の調節経路に重要である。最もよく特徴付け細胞質ゾルRNA結合タンパク質のうち鉄調節タンパク質 、IRP1及びIRP2がある。彼らは、それによってmRNAの翻訳や安定性​​を制御する、いくつかの標的mRNAの翻訳領域（UTR）内の応答要素（IRES）を鉄に結合する。 IRE / IRP相互作用は広くEMSAによって研究されている。ここでは、他のRNA結合タンパク質の活性を評価するために一般化することができるIRP1及びIRP2のIRE結合活性を分析するためのEMSAプロトコルを記述する。 RNA結合タンパク質、またはこのタンパク質の精製調製物を含む粗タンパク質溶解物を、複合体形成を可能にする、32 P標識したRNAプローブの過剰と共にインキュベートする。ヘパリン結合プローブする非特異的タンパク質を排除するために添加される。その後、混合物をポリアクリルアミドゲル上で非変性電気泳動によって分析される。遊離プローブRNA /タンパク質複合体の展示遅滞モビリティながら、高速で移動し、。したがって、手順​​は、「ゲル遅延」または「バンドシフト」アッセイと呼ばれている。電気泳動終了後、ゲルを乾燥させ、RNA /タンパク質複合体、ならびに遊離プローブは、オートラジオグラフィーによって検出される。プロトコルの全体的な目標は、IRE / IRP及び他のRNA /タンパク質相互作用を検出および定量化することである。また、EMSAも調査中のRNA /タンパク質相互作用の特異性は、結合親和性、および化学量論を決定するために用いることができる。

プロトコルのセクション3と4で説明したように、放射性標識IREプローブを調製した。プローブの配列は、5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG C UUCAA C AGUGC UUGGA CGGAUCCU-3」だった。太字のヌクレオチドは、重要な特徴であるIRE不対C残基およびループを表す。プローブの比放射能は、RNAの4.5×10 9 CPM /μgのだった。 IRE結合活性に対する鉄の摂動の効...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

ここでは、RNA /タンパク質相互作用を分析するためのプロトコルを提示する。電気泳動移動度シフトアッセイ（EMSA）は、ネイティブゲル電気泳動中のRNA /タンパク質複合体および遊離RNAの差動移動に基づいている。放射性標識RNAプローブを用いて、RNA /タンパク質複合体は、オートラジオグラフィーによって可視化することができる。

IRE / IRP例：RNA  - タンパク質相互作用の研究のための電気泳動移動度シフトアッセイ（EMSA）

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K ビュー.  Jewish General Hospital. ここでは、RNA /タンパク質相互作用を分析するためのプロトコルを提示する。電気泳動移動度シフトアッセイ（EMSA）は、ネイティブゲル電気泳動中のRNA /タンパク質複合体および遊離RNAの差動移動に基づいている。放射性標識RNAプローブを用いて、RNA /タンパク質複合体は、オートラジオグラフィーによって可視化することができる。 

ビデオ: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

血管新生の研究のために最適化されたフィブリンゲルビーズアッセイ

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

コンピュータによって生成された動物モデルの刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

タンパク質-DNA相互作用の研究のための単一分子マニピュレーションとイメージングを組み合わせる

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

機能電気泳動移動度シフトアッセイ（EMSA）を用いた非コーディング遺伝的変異体およびDNA親和性沈殿アッセイ（DAPA）のスクリーニング

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...