この研究は、ISF、NSFとERCによってサポートされていました。我々はランディミルフォード、マイケル·コーレン、ダグ·シェーファー、ジェレミー·デニソンから温度段階で技術的なヘルプを承認したいと思います。ソフトウェア開発と援助はまた、陳ディ徐ラジェッシュSannareddy、とスミットBhattacharyによって提供されました。我々 は 、MPのIBPタンパク質と有用な議論を私たちの共同研究者教授ピーターL.デービス博士とローリーA.グラハムに感謝したいと思います。また、研究室のメンバードクターマヤバーDolev、Yangzhong秦博士Yelizチェリック博士ナタリアPertaya、Ortal Mizrahy、およびそれらのユーザーからのフィードバックのためのガイShlomitに感謝します。

0。予備手続き<ol><li> ガラスが溶液注入用キャピラリー。キャピラリープラー（ナリシゲ、東京、日本）を用いて、ガラスキャピラリーマイクロチューブから微小開口（ブランド社、ヴェルトハイム、ドイツ）を鋭いピペットを準備します。開口部の大きさは、きれいな水で細かい泡立ちを取得するために毛細血管を介して空気を通過させることによって検証する必要があります。毛細血管が閉じているなら、1つはその縁を壊すことによって、それを開くことができます。これは、管壁を含む水に対して軽く押したり傷ついたりすることによって達成することができる。開口部はほぼ遮断されますが、サブミリメートルの気泡の形成を可能にするために十分に開放されていることをキャピラリーなどを準備します。 </li><li> 銅ディスクのクリーニングは。再蒸留水で洗浄した後、0.1％マイクロ-90石鹸（コール-パーマー、ヴァーノンヒルズ、イリノイ州、米国）で10分間銅ディスクを超音波洗浄します。イソプロパノール（技術）ソリューションにディスクを導入し、10分間超音波処理し、再び。フィン同盟国、ろ過された空気を使用してディスクを乾かしてください。このクリーニングステージは実験間IBP汚染を避けるために重要です。 </li><li> ダブルレイヤーカバーガラスアセンブリ。カバーガラスアセンブリは、カバーガラスの表面に水分を凝縮させずに試料の観察を可能にするために調製した。これは、ホットグルーガンで接着したカバーガラスの間に2 DRIERITE（ワシントン州ハモンドDRIERITE、クセニア、オハイオ州、米国）粒子（直径2mm）を置くことによって達成された。この構成では、サンプルを低温に冷却し、観察窓の上に乾燥した空気を爆破する必要があると削除されたときに視界を遮る可能性が結露を防止。 </li></ol> 1。冷却ステージセットアップ<ol><li> 4ミリメートル、内径タイゴンチューブ（サンゴバン社、パリ、フランス）への冷却段階の水流入口と出口を接続し、水ポンプへの水の流入口チューブを接続します。 </li><li>冷却ステージトンの入口に4ミリメートル内径タイゴンチューブを接続乾燥した空気をお届け​​O。空気はインラインDRIERITEカラムを用いて乾燥した。 </li><li>空気や水ポンプを操作してください。冷却素子はヒートシンクなしで実行されるべきではないことに注意してください。 </li><li>温度調節器、カメラ、およびLabVIEWのルーチンをオンにします。 </li></ol> 2。試料調製<ol><li>ディスクを介して掘削さ500μmの孔を有する直径7mmの銅ディスクの裏側に浸油B（Cargille研究所、シーダーグローブ、ニュージャージー州、米国）の3-4液滴を配置します。 </li><li>下向きにイマージョンオイル側と冷却ステージ上の銅ディスクを置きます。 </li><li> 2ミリリットルガラスシリンジ（Poulten·グラーフ、ヴェルトハイム、ドイツ）にもう一方の端に接続さ0.7ミリメートル、内径タイゴンチューブにキャピラリーチューブ（鈍端）を接続します。 </li><li>事前の毛細管を使用した場合と、（予備手順を参照）開口部が適切なサイズであることを保証するために毛細血管の小さな開口部をチェックします。 </li><li>ゆっくりGLAを挿入ガラスキャピラリーが0.1を含むまでssは準備のIBPタンパク質試料管へのキャピラリ（20 mM CaCl 2およびpH8の25mMトリス-HCl、2.4μMのMpが IBP-GFP、準備の詳細については参考文献10を参照）、ガラスシリンジを引くタンパク質溶液の液。 </li><li> LabVIEWソフトウェアを経由してビデオの録画を開始します。 </li><li>冷却ステージ上の銅ディスクの穴の1つにガラスキャピラリー（タンパク質溶液を含有する）の鋭いエッジを挿入します。 </li><li> （オリンパス、東京、日本、10倍対物レンズ）顕微鏡で観察しながら、慎重にガラスキャピラリー先端で液浸油の層に浸透し、タンパク質の少量（〜10 NL）を提供するガラスシリンジを（非常に微妙）を押してください200μmの液滴を作成するためのソリューションを提供します。 </li><li>二重層カバーガラスアセンブリ（予備手順を参照してください）​​と冷却ステージに穴をカバーしています。 </li></ol> 3。 THの活性測定<ol><li>プリSS冷房ボタン、-40に温度を℃に設定</li><li>当初、溶液滴は明らかであろう。 -35〜-30℃の中で、典型的には、低温度、℃で、液滴の色が変わり、溶液が凍結されたことを示す。サンプルは凍結した直後に大量の氷が溶け始めるまで、ゆっくりと温度を上昇させる。温度が徐々に増加し、サンプルを完全に溶融する可能性があり、温度のオーバーシュートを避けるために必要である。 </li><li> 50倍の対物レンズに切り替えて温度を調整することで氷を溶かし始める。この調整は、対話形式で行われ、最終的なステップは、通常、0.002℃の小さい温度ステップを使用して実行され単結晶が残るまで溶融し続けています。結晶の最終的なサイズは10μm前後となるはずです。融解が消滅したた最高温度は融点であると判断され、後にビデオ分析の段階で正確に決定されます。 </li> &lt;LI&gt;結晶の融点以下の摂氏の数百分のに温度を設定し、10分遅れとの温度勾配を開始します。必要に応じて、ランピング速度を調整します。この間、結晶がIBPSにさらされることになる。 </li><li> 10分間の露光時間が終了すると、温度は、LabVIEWルーチンの制御下で自動的に減少します。 </li><li>温度が下がるなどの結晶形を観察します。ある時点で、氷晶の突然のバーストが観察されることがあります。これが起こる温度は、結晶のバースト温度として注目されている。 </li><li>正確な融点とバーストの温度を決定するために、ビデオ分析を使用しています。まず、ビデオ分析を使用することにより、正確な融点を見つける。融解が消滅したた最高温度は融点であると判断されたことを思い出してください。スプレッドシートプログラムでこの融点を文書化します。その後、正確な結晶バースト温度を決定し、この値を記録同様に。融点と凝固点の差、または水晶バースト温度は、IBPの溶液の熱ヒステリシス活性である。 </li></ol> 4。時間依存のTH活性の測定<ol><li>氷の単結晶を準備するためにセクション3.1から3.3で説明されたプロトコルに従ってください。 </li><li>結晶を形成した後、必要に応じてランプの遅延時間を設定して、ランプをオンにします。 </li><li>温度は自動的に一度ランプ遅延時間が経過した（事業者の要件に応じて）一定の割合で減少します。 </li><li>結晶のバーストが起こる温度を文書化します。露光時間を（結晶形成と結晶バースト間の時間）を計算します。 </li><li>様々な遅延時間の実験を繰り返し、TH活性の時間依存性を評価するために露光時間の関数としてのTH活性をプロットします。 </li></ol>

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特別な利害関係は宣言されません。

この作品は、特別な温度制御とTH活動の正確な測定を可能にするコンピュータ制御によるナノリットル浸透圧計の動作を示しています。任意の温度に敏感なシステムでは、不要な温度勾配は避けなければならない。ここで提示装置内の温度勾配を避けるために、試験液滴が銅ディスク冷却ステージ（ステップ2.7）の穴の中央に配置しなければならない。さらに、単結晶は、液滴の中心ではなく、エッジ付近である必要があります（ほとんどの場合、これは自発的に起こります）。説明時間依存性は、冷却速度がTHの測定値に影響を与える可能性があることを示しています。したがって、私たちは、結晶が冷却する前に溶液に暴露された時間のレポートと同様に、冷却速度を含め示唆している。我々は、典型的には0.01で温度をランプダウンする前に、10分を待っていた℃はそれぞれ4秒を繰り返します。 LabVIEWで制御された共olingステージは、マイクロ流体デバイスは熱的に操作することができた上で倒立顕微鏡で使用するために適応されました。このシステムは、eGFPの9、10、16のタグが付いた氷の結晶とIBPSを含む溶液交 ​​換実験の性能を容易にします。 LabVIEWで制御されたシステムは、指定された適応電気回路を経由して3040温度コントローラを接続することにより、クリフトン·ステージに適合させることができる。このようなシステムは、デイビスラボ17に運営されています。 LabVIEWソフトウェアとクリフトン·ステージのための指定適応電気回路設計のリクエストも承ります。 結論として、我々は温度とリアルタイム解析用のLabVIEWルーチンを介してビデオ·インターフェースとの間で調整温度上昇と減少（0.002℃感度を持つ）、速度の敏感な制御および操作を容易にナノリットル浸透圧計を説明します。このシステムはimportanアール再現レート制御実験を行うことができます氷と一緒にIBPの相互作用の動態を調査するためのトン。このような実験はIBPSの作用機序を取り巻くいくつかの長い議論の問題に対処することができます。

<table border="1"><tbody><tr><td> 名前 </td><td> 会社 </td><td> カタログ番号/モデル </td><td> 注釈 </td></tr><tr><td>イマージョンオイルタイプB </td><td> Cargille研究所</td><td> 16484 </td><td></td></tr><tr><td> DRIERITE </td><td>ワシントン州ハモンドDRIERITE </td><td> 043063 2270グラム</td><td></td></tr><tr><td>マイクロ90洗浄液</td><td>コー​​ル - パーマー</td><td> <a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18100から11</a> </td><td></td></tr><tr><td>プーラーをキャピラリ</td><td>ナリシゲ</td><td> PB-7 </td><td></td></tr><tr><td>ガラス毛細管</td><td>ブランドGNBH </td><td> 7493 21 </td><td>長い75ミリメートル、直径1.15 </td></tr><tr><td>温度コントローラ</td><td>ニューポート、アーバイン、カリフォルニア州、アメリカ合衆国</td><td>モデル3040 </td><td>モデル3040 </td></tr><tr><td>光学顕微鏡</td><td>オリンポス</td><td>モデルBH2 </td><td></td></tr><tr><td> 10倍の対物レンズ</td><td>オリンポス</td><td></td><td> Sプラン10、0.3、160/0.17 </td></tr><tr><td> 50倍の対物レンズ</td><td>ニコン</td><td></td><td> CFの計画、50X/0.55 EPI ELWD </td></tr><tr><td> CCDカメラ</td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td>タイゴンチューブ</td><td>サンゴバン、パリ、フランス</td><td></td><td>タイゴン製剤S-50-HLチューブ</td></tr><tr><td>ガラス注射器（2ミリリットル） </td><td> Poulten·グラーフ、ヴェルトハイム、ドイツ</td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-PCIカード</td><td>ナショナルインスツルメンツでは、米国テキサス州オースティン</td><td> 778032から01 </td><td></td></tr><tr><td>ビデオフレームGRAbber IMAQ PCI-1407 </td><td>ナショナルインスツルメンツでは、米国テキサス州オースティン</td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEWシステム設計ソフトウェア</td><td>ナショナルインスツルメンツでは、米国テキサス州オースティン</td><td>バージョン8 </td><td></td></tr><tr><td> DivXの著者ソフトウェア</td><td> DIVX LLCは、サンディエゴ、CA、USA </td><td></td><td></td></tr></tbody></table>

labview-operated novel nanoliter osmometer for ice binding protein investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

THの時間依存性の測定 LabVIEWのオペナノリットル浸透圧計は、正確なTHの活性測定の性能を容易にします。一定温度の低減率は、TH時間依存性の測定を可能にした。ナノリットル浸透圧計では有効になって正確な温度制御は、これらの実験のために重要だった。溶液中IBPSに氷晶の露光時間は、水晶（クリスタルバースト）の周りに氷の急激な成長まで、結晶の形成からの時間（溶融プロセスの終了）として定義されています。我々はIBPSに氷の結晶の露光時間が決定的にTHの活動に影響を与えたことがわかった。 IBPの露出（数秒）の短い期間では 、MP IBP-GFPの溶液（2.4μM）（ 図5）の低THの活動を作り出した。それは4分のIBP露出でプラトーに達するまでのTH活動はIBP露光時間とともに増加した。高いIBPの濃度で、プレートauのは、短い時間に達しました。 <img alt="図1" src="/files/ftp_upload/4189/4189fig1.jpg" /> 図1：氷に吸着IBPSを示す模式図。 10の許可を得て採択された。 <img alt="図2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> 図2冷却ステージ。 A）は顕微鏡のチューブに接続されています。 B）の上側のリード線なし。 C）の模式図。 <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="図3" src="/files/ftp_upload/4189/4189fig3.jpg" /> 図3。LabVIEWインタフェースのスクリーンショット。 <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">塩素</a>拡大図を表示するには、ここをICK。 <img alt="図4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> 図4。温度安定性のグラフ。温度コントローラは、温度0.01℃毎15秒を下げるために設定されていました。 <img alt="図5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> 図5 IBPSに氷結晶露光時間の関数として mp IBP THの活動。それぞれの時点では、3月6日の実験の平均値である。

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

また、不凍タンパク質として知られている氷結合タンパク質（IBPS）は、氷の成長を阻害し、組織の凍結保存に使用するための有望な添加剤です。 IBPSを調査するために使用する主なツールは、ナノリットル浸透圧計です。我々は、光学顕微鏡に取り付けられた家に設計された冷却ステージを開発し、特注のLabVIEWのルーチンを使用して制御する。ナノリットル浸透圧計は、超高感度の方法で試料温度を操作ここで説明する。

アイス結合タンパク質の調査用のLabVIEWオペ新規ナノリッター浸透圧計

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

Research

JoVE Journal

Chemistry

20.3K ビュー.  The Hebrew University of Jerusalem. また、不凍タンパク質として知られている氷結合タンパク質（IBPS）は、氷の成長を阻害し、組織の凍結保存に使用するための有望な添加剤です。 IBPSを調査するために使用する主なツールは、ナノリットル浸透圧計です。我々は、光学顕微鏡に取り付けられた家に設計された冷却ステージを開発し、特注のLabVIEWのルーチンを使用して制御する。ナノリッ?...

ビデオ: LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

単一分子タンパク質研究のための表面パッシベーション

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

化学

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

共有結合は、自己組織化単分子膜の手法を用いて表面上のBMP-2の結合

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

蛍光ベースの氷面アフィニティーによる不凍タンパク質の氷結合平面の決定

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

タンパク質 - タンパク質相互作用およびアロステリックリガンド効果の反応速度を測定するためのバイオレイヤー干渉法

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

マラリアのタンパク質バイオマーカーヒスチジンリッチプロテイン-IIの検出のためのイリジウム（III）発光プローブ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

油中水型エマルジョン：疎水性顔料と水溶性クロロフィル結合タンパク質を組み立てるための新しいシステム

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

ELISAベースの結合と競争法は急速にリガンド - 受容体相互作用を決定するために、

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE  - クリア天然ゲル溶出液フラクションエントラップメント電気泳動

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

EOB-DTPAとそのののGa（III）錯体に関する研究<sup&gt; 68</sup&gt; Gaの放射性標識アナログ

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

光応答性液晶の構造ダイナミクスを観察する手法

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...