この研究は、国立衛生研究所の国立心臓・肺・血液研究所の壁内研究部門によって支援されました。S.H.は日本の官民パートナーシップ学生留学プログラムの支援を受け、H.N.とK.Y.は武勇とVドラッグ海外研修奨学金を受けた。

1. SDS-ページとPVDF膜への転送
<ol><li>相対サイズに基づいてタンパク質を分離するためにSDS-PAGEを行います。 注:商用または自家製のゲルシステムは問題ありません。製造元のプロトコルに従ってください。</li>
	<li>分離したタンパク質をゲルからPVDF膜に移す。 注:一般的な転送方法(半乾式または湿式転送)が適しています。製造元のプロトコルに従ってください。</li>
</ol>2. PVDF膜の遮断
<ol><li>膜を室温で撹拌して1時間適切なブロッキングバッファーにインキュベートします。 注:一次抗体および検出システムに応じて、適切なブロッキングバッファを選択する必要があります。例えば、最も典型的なブロッカーはBSA、非脂肪乾燥乳、カゼイン、および市販の合成ポリマーである。Tween20 が 0.1% の PBS および TBS は、最も一般的に使用されるバッファーです。このブロッキング工程は、4°Cで一晩行なわれてもよい。</li>
</ol>3. 一次抗体によるインキュベーション
<ol><li>蒸留水で10%免疫反応増強剤-1溶液(IRE-1)を調製する。渦はよく。</li>
	<li>10%IRE-1で希釈された一次抗体を調製します。やさしく、十分に混ぜます。</li>
	<li>より大きな膜(4 cm x 8 cm - 8 cm x 8 cm)を使用する場合は、抗体溶液の8 mLを50 mL円錐形遠心分離管に加えます。より小さい膜(2 cm x 8 cm - 4 cm x 8 cm)を使用する場合、抗体溶液の3 mLを14 mLの丸底ポリプロピレンチューブ(材料表)に加える。</li>
	<li>ピンセットでPVDF膜を拾い、ブロッキング溶液を簡単に排出します。PVDF膜を手袋をした指でこのチューブに挿入し、膜全体がチューブの壁に付着していることを確認します。PVDF膜をチューブに挿入すると、もともとゲルに接触していた膜の「タンパク質側」に向かって、内側に向かいます。キャップをしっかりと閉じます。</li>
	<li>50 mLチューブをガラス瓶に入れます。 注:このインキュベーションに14 mLチューブを使用する場合は、50 mLチューブの中央にインナーチューブを保持するためにプラスチックリングホルダーのペアを使用して、50 mLチューブに14 mLチューブを挿入します。</li>
	<li>ハイブリダイゼーションオーブンをオンにします。</li>
	<li>少なくとも5分間、6rpm回転で膜をインキュベートする。 注:膜が常に壁に付着していること、および(内側の)チューブが抗体溶液で膜を均等に覆うために水平であることを確認してください。実際の実験の前に抗体の希釈とインキュベーション時間を較正します。</li>
</ol>4. 膜洗浄
<ol><li>回転を停止します。</li>
	<li>ピンセットでチューブからPVDF膜を慎重に取り出し、50 mLのPBS-Tの容器に入れます。</li>
	<li>膜をPBS-Tで短時間すすいでください。</li>
	<li>気泡が出なくなるまで、蒸留水で容器内の膜をすすいでください。これは抗体の大部分を除去する。</li>
	<li>PVDF膜を容器から250 mLのPBS-Tを含むサラダスピナーに移します。</li>
	<li>ストレーナーバスケットをスピナーに入れます。蓋がしっかりと付けられていたことを確認します。</li>
	<li>スピナーをアクティブにし、約20〜30 sでそれを実行します。</li>
	<li>溶液を捨て、蒸留水でスピナーの内側を簡単にすすきます。</li>
	<li>250 mL の PBS-T を再度スピナーに加えます。</li>
	<li>手順 4.6 と 4.7 を繰り返します。</li>
</ol>5. 二次抗体を用いてインキュベーション
<ol><li>蒸留水で10%免疫反応増強剤-2溶液(IRE-2)を調製する。渦はよく。</li>
	<li>10%IRE-2で希釈された二次抗体を調製します。やさしく、十分に混ぜます。</li>
	<li>ステップ3.3に記載されている抗体溶液およびチューブの体積を選択します。</li>
	<li>ピンセットでPVDF膜を拾い、溶液を簡単に排出します。手順 3.4 に記載されているように、PVDF 膜をチューブに挿入します。</li>
	<li>50 mLチューブをガラス瓶に入れます。</li>
	<li>ハイブリダイゼーションオーブンをオンにします。</li>
	<li>少なくとも5分間、6rpm回転で膜をインキュベートする。膜が常に壁に付着していること、および(内側の)チューブが抗体溶液で膜を均等に覆うために水平であることを確認してください。 注: 実際の実験の前にインキュベーション時間を調整します。二次抗体が蛍光結合されると、暗所でインキュベーションを行う。</li>
</ol>6. 膜洗浄
<ol><li>手順 4.1 ~ 4.10 に従います。</li>
</ol>7. 画像の取得
<ol><li>化学発光検出<ol>
<li>半移植柔軟フィルム(10 cm x 15 cm)を平らな表面に置きます。</li>
		<li>5 mL チューブに化学発光基材の 2 つの成分を混合します。</li>
		<li>混合基材の1.5mLを半移植柔軟なフィルムに直ちに移し、PVDF膜をその上に置きます。次いで、残りの基質溶液を膜上に移す。</li>
		<li>半透明のフレキシブルフィルム上に混合基材を用いてPVDF膜を1分間インキュベートします。</li>
		<li>ピンセットでPVDF膜をピックアップし、基質溶液を簡単に排出します。</li>
		<li>透明フィルムのペアの間に膜を挟みます。</li>
		<li>化学発光モードで画像を取得します。</li>
	</ol>
</li>
	<li>蛍光検出<ol>
<li>ピンセットでPVDF膜を拾い、溶液を簡単に排出します。</li>
		<li>透明フィルムのペアの間に膜を挟みます。</li>
		<li>蛍光モードで画像を取得します。 メモ:ステップ7.1と7.2は、最大感度を確保するために、イメージングマシンに近接して実行する必要があります。</li>
		<li>バックグラウンドシグナルが高い場合、PBS-Tの80〜100 mLの容器内で洗浄工程を行い、10分リンスを一次抗体に対して3回、2次抗体に対して5分リンスを6回行う。 注:10%のIRE溶液で希釈された一次抗体と二次抗体の両方を、感度6を失うことなく最大8〜10回使用できます。溶液は、最大1ヶ月間4°Cに保つ必要があります。</li>
	</ol>
</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+critical+factors+for+quantitative+immunoblotting.">An analysis of critical factors for quantitative immunoblotting.</a> Science Signaling. 8 (371), (2015).</li><li>Schuck, P., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+transport+limitation+and+surface+heterogeneity+in+the+biophysical+characterization+of+macromolecular+binding+processes+by+SPR+biosensing.">The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.</a> Methods in Molecular Biology. 627, 15-54 (2010).</li><li>Li, J., Zrazhevskiy, P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Size-Associated+Diffusion+Constraints+for+Rapid+On-Surface+Bioassays+with+Nanoparticle+Probes.">Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.</a> Small. 12 (8), 1035-1043 (2016).</li><li>Higashi, S. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. 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ウェスタンブロットは、約40年前に開発された特定のタンパク質を検出するために広く使用されている分析技術です7,8.それ以来、技術は進化し続けており、その後の技術革新により、技術2、9、10、11、12、13の感度、速度?...

ウェスタンブロット(イムノブロットとも呼ばれる)は、幅広い科学的および臨床的分野にわたる強力で基本的な技術です。この技術は、タンパク質1の存在、相対的存在量、相対分子質量、および翻訳後修飾を調べるために使用される。デジタル画像解析と組み合わせることで、この方法はタンパク質およびタンパク質修飾の豊富さを確実に分析することができます2.ウェスタンブロットは日常的に行われますが、時間と労力を要する方法です。膜を有する抗体の長いインキュベーションが必要です。ここでは、感度を犠牲にすることなくこの制限を克服するインキュベーション法の改変について述べる。
膜のインキュベーション中、抗体は溶液中に浮遊し、抗原は膜上に固定化される。その高い親和性のために、抗体が抗原に結合する速度は、バルク溶液から膜への抗体の拡散よりも速い。これにより、低濃度の「枯渇層」が作成されます(図1)。この技術では、より遠い抗体が受動的拡散によって膜に到達するまでに数時間かかることがあります。この効果は、大量輸送制限(MTL)3と呼ばれます。抗体含有溶液の繰り返しの排水と補充は、枯渇層を破壊し、MTL4を克服する可能性が提案されている。ここでは、従来のウェスタンブロッティングプロトコルにおけるMTLの効果を減少させ、必要な潜伏期間を顕著に短縮するために、この循環ドレインおよび補充(CDR)概念を実装する独自の技術を考案しました。
短時間で検出感度を維持するために、抗原抗体反応を加速する免疫反応増強技術を用いて、シグナル対ノイズ比5を向上させた。市販の免疫反応増強剤(IRE)の多くは2成分から構成されている(溶液1および2、表の材料表参照)。IREの独自の組成または作用機序は明らかにされていないが、固相結合アッセイにおいてIREが抗原と抗体との間の解離定数を低下させることを以前に発見したが、少なくとも部分的には、IRE6の増強効果を担う親和性の増加を示す。CDRとIREの組み合わせは、感度を犠牲にすることなく、手順全体の時間を短縮する超高速ウェスタンブロッティングプロトコルを生み出します。

<table>
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td>
			<td>Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Apollo Transparency Film for Laser Printers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any kinds of Laser Printer Transparency Film are fine.</td>
		</tr>
		<tr>
			<td>Azure Imaging System 600</td>
			<td>Azure Biosystems</td>
			<td>Azure 600</td>
			<td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td>
		</tr>
		<tr>
			<td>Can Get Signal Immunoreaction Enhancer Solution</td>
			<td>TOYOBO</td>
			<td>NKB-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CARNATION Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from sheep</td>
			<td>GE Healthcare</td>
			<td>NA9310V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from donkey</td>
			<td>GE Healthcare</td>
			<td>NA9340V</td>
			<td></td>
		</tr>
		<tr>
			<td>HYBAID Micro-4</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane, 0.45 &#181;m pore size</td>
			<td>Millipore Sigma</td>
			<td>IPVH304F0</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-32211</td>
			<td></td>
		</tr>
		<tr>
			<td>KPL LumiGLO Reserve Chemiluminescent Substrate</td>
			<td>seracare</td>
			<td>5430-0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#223; actin antibody</td>
			<td>Millipore Sigma</td>
			<td>A5316</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (PBS)</td>
			<td>LICOR</td>
			<td>927-40100</td>
			<td>Blocking Buffer for fluorescent detection</td>
		</tr>
		<tr>
			<td>OXO Salad Spinner</td>
			<td>OXO</td>
			<td>32480V2B</td>
			<td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td>
		</tr>
		<tr>
			<td>Parafilm Sealing Film</td>
			<td>Bemis</td>
			<td>Parafilm M PM996</td>
			<td>semi-transparent flexible film</td>
		</tr>
		<tr>
			<td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Rings to hold 14 ml tube in the center of 50 ml tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared in a machine shop</td>
		</tr>
		<tr>
			<td>Rabbit anti-6X His tag antibody</td>
			<td>Abcam</td>
			<td>ab9108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultra-high-speed western blot using immunoreaction enhancing technology

ウェスタンブロット(免疫ブロットとも呼ばれる)は、生物医学研究のための正規の方法です。これは、特定のタンパク質の相対的なサイズと存在量だけでなく、翻訳後のタンパク質修飾を決定するために一般的に使用されます。この技術は豊かな歴史を持ち、そのシンプルさのために広く使用されています。しかし、ウェスタンブロッティング手順は、スループットを制限する長いインキュベーション時間であるという重大なボトルネックで、完了までに数時間、さらには数日かかることで有名です。これらのインキュベーションステップは、バルク溶液から膜上の固定化された抗原への抗体の拡散が遅いために必要とされる:膜付近の抗体濃度はバルク濃度よりもはるかに低い。ここでは、抗体溶液の循環ドレインおよび補充(CDR)による抗原結合を改善することにより、これらのインキュベーション間隔を劇的に短縮するイノベーションを提示する。また、免疫反応増強技術を用いて、アッセイの感度を維持しました。市販の免疫反応増強剤とのCDR法の組合せは、出力シグナルを高め、抗体インキュベーション時間を実質的に短縮した。得られた超高速のウェスタンブロットは感受性の損失なしで20分で達成することができる。この方法は、化学発光と蛍光検出の両方を使用してウェスタンブロットに適用することができます。この簡単なプロトコルにより、研究者は多くのサンプルでタンパク質発現の分析をよりよく探求することができます。

この例は、ウェスタンブロット上の免疫増強技術と共にCDR法の有効性を示す。スタティックインキュベーションは、PVDF膜を抗体溶液と共にインキュベートした半透明のフレキシブルフィルムに対して行ったのに対し、CDRインキュベーションは膜および抗体溶液を含む回転チューブによるハイブリダイゼーションオーブン中であった(Movie)。 図2 は、ウェスタンブロット?...

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Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

超高速ウェスタンブロッティング技術は、免疫反応促進剤と組み合わせて、環状ドレインおよび補充(CDR)技術を介して抗原抗体結合の運動を改善することによって開発される。

免疫反応向上技術を用いた超高速ウェスタンブロット

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

5.8K ビュー.  National Institutes of Health. 超高速ウェスタンブロッティング技術は、免疫反応促進剤と組み合わせて、環状ドレインおよび補充(CDR)技術を介して抗原抗体結合の運動を改善することによって開発される。

ビデオ: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

生化学

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

西部のしみが付くプロトコル造血幹細胞の数が少ない

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

高スループット、定量的なドットを使用して組織レベルで選択されたタンパク質の含有量の絶対測定のしみの分析 (おっ)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Studying Protein Import into Chloroplasts Using Protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

プロトプ ラストを用いた葉緑体への蛋白質輸送を勉強

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

コアラでのコルチゾール測定(ファスコラルコトス・シネレウス)毛皮

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

タンパク質レベルの組織や標準化された定量ウェスタンブロットによる開発全体の堅牢な比較

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

アフィニティクロマトグラフィーによるインノシトールリン酸塩またはホスホイノシチド相互作用タンパク質の同定

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

植物マイクロRNAの検出と定量のためのRNAブロット分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

単粒子低温電子顕微鏡用生体試料の手動ブロットとプランジ凍結

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

電界効果バイオセンシング技術を用いた分子シャペロンHsp90とそのクライアントプロテインキナーゼCdc37との生体分子相互作用の探索

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...