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>

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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&amp;rsquo;)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&amp;rsquo;)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 &amp;micro;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-&amp;szlig; 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?...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

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

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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

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

赤外蛍光色素で標識されたオリゴヌクレオチドを使用したゲルシフトアッセイのために最適化されたプロトコル

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

生化学

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

西洋は、Invitrogen NuPage NOVEXビストリスミニゲルを用いたウェスタンブロット

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

生物学

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

タンパク質キャピラリー等電点焦点法によるアイソ フォームの高感度・定量的検出

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

イムノブロット解析

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

ウェスタンブロッティング：サンプル調製を検出する

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

町の西部最速：クラシックウェスタンブロット分析に現代風にアレンジ

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

免疫沈殿ベースの技術:アガロースビーズを用いた内因性タンパク質の精製

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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高等生物学

免疫学

The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

ウエスタンブロット法

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

基礎生物学

細胞および分子生物学の基本的方法

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app.jove.com/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

タンパク質プロセッシングのための化学発光ウェスタンブロットアッセイ

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...

免疫反応向上技術を用いた超高速ウェスタンブロット

要約

さらに動画を探す

この動画の章

赤外蛍光色素で標識されたオリゴヌクレオチドを使用したゲルシフトアッセイのために最適化されたプロトコル

西洋は、Invitrogen NuPage NOVEXビストリスミニゲルを用いたウェスタンブロット

タンパク質キャピラリー等電点焦点法によるアイソフォームの高感度・定量的検出

イムノブロット解析

ウェスタンブロッティング：サンプル調製を検出する

町の西部最速：クラシックウェスタンブロット分析に現代風にアレンジ

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

免疫沈殿ベースの技術:アガロースビーズを用いた内因性タンパク質の精製

ウエスタンブロット法

タンパク質プロセッシングのための化学発光ウェスタンブロットアッセイ

免疫反応向上技術を用いた超高速ウェスタンブロット

要約

さらに動画を探す

この動画の章

赤外蛍光色素で標識されたオリゴヌクレオチドを使用したゲルシフトアッセイのために最適化されたプロトコル

西洋は、Invitrogen NuPage NOVEXビストリスミニゲルを用いたウェスタンブロット

タンパク質キャピラリー等電点焦点法によるアイソ フォームの高感度・定量的検出

イムノブロット解析

ウェスタンブロッティング：サンプル調製を検出する

町の西部最速：クラシックウェスタンブロット分析に現代風にアレンジ

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

免疫沈殿ベースの技術:アガロースビーズを用いた内因性タンパク質の精製

ウエスタンブロット法

タンパク質プロセッシングのための化学発光ウェスタンブロットアッセイ

タンパク質キャピラリー等電点焦点法によるアイソフォームの高感度・定量的検出