著者らは、AHに生命​​賞のUF科学ハワードヒューズ医学研究所に感謝

1。カセットの準備<ol><li>クリーン前面と背面に使用インビトロジェンNupage NOVEXミニゲルカセット、またはその他の商業ミニゲルカセットのプレート。 </li><li>プラスチックエポキシの約1-3 mlを準備し、製造元の指示に従って、よく混ぜます。 </li><li>カセットがアセンブルされたときに2つのプレートが接触する前面板の内側にエポキシ樹脂を適用します。 </li><li>開口部を封止するから余分なエポキシを防ぐために、カセットを組み立てるときにバックプレートの下にスリットを通過フィルタ紙や段ボールの部分をスライドさせます。とすぐに二つのプレートが組み立てられるようにろ紙を削除します。 </li><li>密封のためのエッジに沿ってバインダークリップを配置し、カセットが完全に硬化するエポキシ樹脂のために一晩放置。 </li><li>電気テープとバックプレートの底部にスリットを密封する。 </li></ol> 2。分離ゲルを注ぐ<ol><li> 125mlのサイドアームフラスコに、次の行を追加します。4Xトリス·ベース分離ゲル緩衝液8.70、30.8％アクリルアミド/ビスアクリルアミドの3.50ミリリットルの2.25ミリリットル、9ミリリットルの合計ボリュームのタイプI試薬グレード水（IRG-H 2 O）の3.25ミリリットル。 </li><li>脱ガス、真空装置にフラスコのサイドアームを取り付け、フラスコの上に反転したストッパーを配置することによって、10分間のソリューションを提供します。 </li><li> 50ミリリットル使い捨てのポリプロピレン製遠心管に脱ガス処刑され、ソリューションの9ミリリットルを転送し、30 10％のammounium過硫酸アンモニウム（APS）の添加およびNμlの6、N、N &#39;、N&#39;、 - テトラメチルエチレンジアミン（TEMED）とミックスを追加する気泡を導入することを避けるためのソリューションを渦巻くによっても。 </li><li>すぐにそれはゲルプレートの間に形成される任意のバブルを防ぐために、カセットの側面を下に実行することができ、カセットに溶液を注ぐ。フロント（短い）プレートの上端から約2 cmにカセットを入れます。 </li><li>優しく1X分離ゲル用緩衝液で飽和1 - ブタノール0.5mlを追加し、ゲルが1の重合せ時間。 </li></ol> 3。スタッキングゲルを注ぐ<ol><li> 4X Tris塩基スタッキングゲル緩衝液pH 6.80、30.8％アクリルアミド/ビスアクリルアミドの0.40ミリリットル、3ミリリットルの合計ボリュームのIRG-H 2 Oの1.85ミリリットルの0.75ミリリットル：125 mlのサイドアームフラスコに以下を追加します。 </li><li>脱ガス、ステップ2で説明したように10分間のソリューションを提供します。 </li><li>スタッキングゲル溶液は脱気されていますが、分離ゲルの上端から1X分離ゲル用緩衝液で飽和1 - ブタノールをオフに注ぐ。 IRG-H 2 Oで一回分離ゲルの上部をすすぐ水を注ぎ、残りの液体を吸収する吸湿性またはワットマンろ紙を使用しています。 </li><li>あなたの脱気した溶液3mlを削除し、10％APSとTEMED 2μlの9μlを加え、よく混ぜる。 </li><li>迅速に、より短いフロントゲルプレートの上端付近になるまでは、カセットの側を下にして実行することができ、カセット溶液を注ぐ。 </li><li> 、ゲルカセットの上部にきれいなゲルコームを挿入します。所定の位置に保持するために櫛を介してカセットにバインダークリップを配置します。スタッキングゲルは一晩に1時間重合してみましょう。 </li></ol> 4。ゲルを保存する<ol><li>ゲルが完全に重合した場合には、バインダークリップを削除して、ヒートシール可能なプラスチック製の袋の中にカセット、または再シール可能なプラスチック袋を置きます。 </li><li>ポーチに0.1％アジ化ナトリウムが含まれている1Xスタッキングゲルバッファー20mlを注ぐ。 </li><li>熱が5秒のために袋を密封して袋に入れて漏れがないことを確認してください。 </li><li> 4℃でゲルを保存する</li></ol> 5。サンプルの準備<ol><li>薄肉のマイクロ遠心チューブ、タンパク質試料の6.5μlにまで追加して、2倍のNOVEXトリス-グリシンSDSサンプルバッファー7.5μL、100 mMジチオスレイトール（DTT）およびIRG-H 2 O（必要に応じて）を1μlで15μlの合計体積。 </li><li>ボルテックスによりサンプルを混合し、簡単にチューブの底に内容を持って来るためにサンプルを遠心します。 </li> &lt;LI&gt;変性70℃サンプル℃で10分間。 </li></ol> 6。ゲルを実行する<ol><li> NOVEXゲル装置を設定します。 </li><li>内室へNupage酸化防止剤を500μl、及びトリス - グリシンが外側室にバッファを実行している300mlのが含まれているトリス - グリシン泳動緩衝液200 mlを追加します。 </li><li>ゲルローディングピペットチップを使用してウェルに変性したタンパク質サンプル（15μl）をロードします。 </li><li> 60から70分間、200Vでゲルを実行します。 </li></ol> 7。ゲルを染色<ol><li>ゲル装置からカセットを取り出して、離れてゲルナイフや硬いパテナイフを使用して、2つのプラスチック製のゲルプレートを詮索好きで、エポキシシールを破る。ゲルを破れないように注意しながら、プレートからゲルを慎重に分離します。 </li><li>穏やかに撹拌しながらIRG-H 2 Oで三回ゲルを洗浄します。 </li><li>穏やかに攪拌しながら1時間インビトロジェンSimplyBlue SafeStainでゲルを染色する。 </li><li>染色液を捨て、IRG-H 2 Oゲルを脱色</li></ol> 8。カセットのクリーニング<ol><li>彼らは再び再利用できるようにカセットにすべてのエポキシこすり落とすか、皮に金属製のヘラを使用しています。 </li><li>中性洗剤溶液をプラスチック製のゲルプレートのそれぞれを洗浄し、蒸留水、続いて水道水でよくすすいでください。 </li></ol> 9。代表的な結果エポキシ樹脂は、防水シール、（図1）を形成するので、カセットはアクリルアミド溶液は、それらに注がれたときにリークされません。さらに、ゲルカセットは簡単に染色ゲルを取得するために開くことができ、エポキシ樹脂は、その継続的な再利用を可能にするためにカセットから剥離することができます。図2に示すように、ゲルは、タンパク質マーカーとルーチンタンパク質の電気泳動に適したサンプルバンドの明確な分離を示しています。 <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="図1" /> 図1。アプリミニゲルカセットを密封するためのエポキシ樹脂のカチオン。エポキシ樹脂の薄層は、従来のアセンブリへのカセットのいずれかのエッジに適用されます。エポキシ樹脂を強調するためには、このイメージの偽色の赤であった。 <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="図2" /> 図2。 SDS-トリス-グリシンゲル上のタンパク質の分離は再使用ミニゲルカセットを使用して注いだ。上記のようにSDS-PAGEを行った。電気泳動後ゲルは、ミニゲルカセットから削除され、製造元の指示に従ってSimplyBlue SafeStainで染色し、送信された白色光を使用して撮影しました。レーンMはSeeBlue Plus2事前に染色された蛋白質の基準が含まれています。レーンM中のタンパク質の分子量はキロダルトン（kDa）のゲルの右側に示されています。他のレーンは、グルタチオン-S-トランスフェラーゼ（GST）タグ付きタンパク質を発現する細菌からの清澄化ライセートからタンパク質を示しています。に示すタンパク質は、それらのgから洗浄されるGST-タグタンパク質の溶出の前にlutathioneビーズ。

<ol>
	<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Raymond, S., Weintraub, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide+gel+as+a+supporting+medium+for+zone+electrophoresis.">Acrylamide gel as a supporting medium for zone electrophoresis.</a> Science. 130, 711-711 (1959).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem. Biophys. Res. Commun. 28, 815-820 (1967).</li></ol>

利害の衝突が宣言されません。

タンパク質のゲル電気泳動は、ほとんどの分子生物学研究所のために不可欠な方法です。市販のミニゲルでは、利便性の高いレベルを提供していますが、コストがかかる場合があります。ここでは、市販のミニゲルの人気ブランドからミニゲルカセットを再利用する方法を示しています。このメソッドは、ゲルの既製のソースを持つことの利便性を保持しますが、市販のミニゲルの数分の一?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>インビトロジェンNupage NOVEXミニゲルカセット（1.0ミリメートル） </td><td>インビトロジェン</td><td> NC2010 </td><td> 2、5、10、12、15、2次元の井戸で利用可能</td></tr><tr><td>プラスチックエポキシ</td><td>ロックタイト</td><td> 108771 </td><td></td></tr><tr><td>電気テープ</td><td>スコットランドの</td><td></td><td></td></tr><tr><td> 125mlのサイドアームフラスコ</td><td>パイレックス</td><td> 5360 </td><td></td></tr><tr><td> 4Xトリス·ベース分離ゲルバッファー</td><td></td><td></td><td> 1.5 M Tris塩基のpH 8.7 </td></tr><tr><td>アクリルアミド</td><td>フィッシャー·サイエンティフィック</td><td> 01065-500 </td><td></td></tr><tr><td>ビスアクリルアミド</td><td> MPバイオメディカル株式会社</td><td> 800173 </td><td></td></tr><tr><td> 30.8％アクリルアミド溶液</td><td></td><td></td><td> 30:0.8アクリルアミド：ビスアクリルアミドIRG-H 2 Oで</td></tr><tr><td> 10％のAPS </td><td>フィッシャー·サイエンティフィック</td><td> BP179-25 </td><td>使用直前に調製した1ミリリットルIRG-H 2 O、0.1 gの過硫酸アンモニウム</td></tr><tr><td> TEMED </td><td>アルコスオーガニック</td><td> 138450500 </td><td></td></tr><tr><td> 1X分離ゲル用緩衝液で飽和1 - ブタノール</td><td></td><td></td><td>午前5時02分1X分離ゲルバッファーに1 - ブタノール</td></tr><tr><td> 4X Tris塩基スタッキングゲルバッファー</td><td></td><td></td><td> 1.5 M Tris塩基のpH 6.8 </td></tr><tr><td>ゲルの櫛</td><td>インビトロジェン</td><td> NC3015 </td><td> 15よく、1.0 mm厚</td></tr><tr><td>インパルスシーラー</td><td> TEW電気暖房機器</td><td></td><td></td></tr><tr><td>ヒートシール可能なポーチ</td><td> Kapak / Scotchpak </td><td></td><td> 1パイントサイズ（6.5 &quot;×8&quot;） </td></tr><tr><td>ゲル格納バッファ</td><td></td><td></td><td> 0.1％アジ化ナトリウムを含む1Xスタッキングゲルバッファー</td></tr><tr><td> NOVEX SDSトリス - グリシンサンプルバッファー（2X） </td><td>インビトロジェン</td><td> LC2676 </td><td></td></tr><tr><td> DTT </td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10XトリスGylcineランニングバッファー</td><td>インビトロジェン</td><td> LC2675 </td><td> 10X株式の1L：29.0グラムのトリスベース、144.0グラムグリシン、10.0グラムSDS </td></tr><tr><td> X細胞SureLockミニ細胞</td><td>インビトロジェン</td><td> EI0001 </td><td></td></tr><tr><td>ゲルナイフ</td><td>インビトロジェン</td><td> EI9010 </td><td></td></tr><tr><td>ステインSimplyBlue安全</td><td>インビトロジェン</td><td> LC6065 </tD&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（SDS-PAGE）分析を用いたタンパク質の評価は、生化学や分子生物学の研究者が1から4で使用される一般的な手法です。タンパク質の毎日の分析を行う研究室では、市販のポリアクリルアミドゲル（〜$ 10/gel）のコストは、時間の経過とともにかなりのことができます。このコストを軽減するために、いくつかの研究者が独自のポリアクリルアミドゲルを準備します。これらのゲルを注ぐの伝統的な方法は、通常、特殊な装置で高価になると多くのゲルを注ぎ、将来の使用のためにそれらを格納し排除することができるガラスゲルプレートを利用しています。また、クリーニングや注入ゲル中のガラスプレートの処理は、学部研究室の授業での使用を排除することが安全上の問題を作成、偶発的破損が発生することがあります。我々のプロトコルは、Invitrogen Nupage NOVEXミニゲルカセットをリサイクルして、同時に複数の蛋白質ゲルを注ぐ方法を示していますし、安価なミリアンペアterialsは、ホームセンターで購入。この経済的かつ合理化された方法は、数週間のために4℃でゲルを保存する方法が含まれています。市販のゲル、頻繁な蛋白質のゲルを実行ラボからプラスチック製のゲルカセットを再利用することで大幅にコストを節約し、環境を助けることができます。さらに、プラスチック製のゲルカセットは、学部研究室の教室のために理想的破損、非常に耐性があります。

Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Pouring and Running a Protein Gel by reusing Commercial Cassettes

我々のプロトコルは、ホームセンターで購入インビトロジェンNupage NOVEXミニゲルカセット、そして安価な材料をリサイクルして、一度に複数のタンパク質ゲルを注ぐ方法を示しています。この経済的かつ合理化された方法は、数週間のために4℃でゲルを保存する方法が含まれています。市販のゲル、頻繁な蛋白質のゲルを実行ラボからプラスチック製のゲルカセットを再利用することで大幅にコストを節約し、環境を助けることができます。

商業カセットを再利用することでタンパク質ゲルを注ぐと実行

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

pouring-and-running-a-protein-gel-by-reusing-commercial-cassettes

Research

JoVE Journal

Biology

24.8K ビュー.  University of Florida. 我々のプロトコルは、ホームセンターで購入インビトロジェンNupage NOVEXミニゲルカセット、そして安価な材料をリサイクルして、一度に複数のタンパク質ゲルを注ぐ方法を示しています。この経済的かつ合理化された方法は、数週間のために4℃でゲルを保存する方法が含まれています。市販のゲル、頻繁な蛋白質のゲルを実行ラボからプラスチック製の?...

ビデオ: Pouring and Running a Protein Gel by reusing Commercial Cassettes

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

セミ変性洗剤-アガロースゲル電気泳動によりアミロイド凝集のためにスクリーニング

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

生物学

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

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

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

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

ポリアクリルアミドゲル電気泳動と銀染色によるグリコサミノグリカンの検出

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

マウスの漸進的レジスタンストレーニングのためのシンプルで安価なランニングホイールモデル

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Modified Matrix Gel Plug Assayによる血管新生研究の簡素化

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

著者スポットライト:Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

血管新生研究のための改変 In Vivo マトリックスゲルプラグアッセイ

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...