この作品は、VFに国立衛生研究所（NS42599とGM077569）からの補助金によって支えられて。

1。架橋の準備<ol><li>あなたは、標準のチューブの反応あたりタンパク質500μgの単離を可能にするためにプレートに細胞の十分な数が必要になります。単管は十分な免疫ブロットによって目的のタンパク質の推定上のインターアクターの識別のためかもしれません。この場合、ビーズ結合した物質は、SDS - PAGEサンプルバッファー（免疫沈降）で溶出することができます。質量分析のために、標準的な反応の数は少なくとも10倍に増加する必要があるとタンパク質の複合体は、（免疫アフィニティークロマトグラフィー）に使用する抗体によって認識されるペプチド抗原と外の競争によって溶出されるべきである。この戦略は、免疫グロブリンの自由なタンパク質複合体の分離が可能になります。 </li><li>架橋のための最適な細胞密度は75％とコンフルエント90％の間です。高コンフルエントな条件で、細胞はすべての細胞への細胞透過性の架橋剤へのアクセシビリティを低下させる、お互いに山積みする傾向がある。 </li><li>細胞の種類や実験条件はセルプレートの底部にラベルする必要があります。我々は日常的に一人で車を持つコントロールが含まれています。 </li><li>前の架橋へのDSPソリューションを除き、すべてのソリューションを準備します。 <ol><li>のCaCl 2 0.1mMの及びCaCl 2実験を開始する前に、1mMの（PBS /カルシウム/マグネシウム）でリン酸緩衝生理食塩水バッファの株式を準備します。カルシウムとマグネシウムイオンの添加は、実験の過程で培養プレートに細胞の接着のための非常に重要です。 4℃で保存します。 </li><li> 50X完全プロテアーゼインヒビターカクテル - 1mLのMilli - Q水に溶解した1錠。 -20℃で保存</li><li> 20％トリトンX - 100 10グラムトリトンX -100を秤量および50 ml Milli - Q水の合計量に希釈する。 4℃ロック4℃一晩と店舗℃で使用するまで。 1カ月以上保存しないでください。以下に説明する溶解およびIPバッファを準備するには、この20％の株式の希釈液を使用してください。 </li><li> pHは7.4〜50倍のDSP焼入れソリューション1Mトリス。室温で保管してください。 </li><li> 10 ×バッファーの溶液を調製します。 10Xバッファー： <ul><li> 50 mLの1 M HEPES </li><li> 150 mLの5 M NaClを</li><li> 10 mLの0.5 M EGTA </li><li> 250μL2 MのMgCl 2 </li><li> 7.4にpHを調整する</li><li> 500mLに、最終容量をもたらす</li></ul> 10X Bufferは解決策は、必要に応じて1Xバッファーに希釈される。 1X Bufferは溶解液と免疫磁気降水量のバッファでも使用されます。 1Xをバッファリング <ul><li> 10mMのHEPES </li><li> 150mMのNaCl </li><li> 1mMのEGTA </li><li> 0.1mMのMgCl 2の </li><li> pH7.4の</li></ul></li><li>溶解バッファー1 ×バッファー+ 0.5％トリトンX - 100。 </li><li>免疫磁気降水バッファ（IPバッファ）1 ×緩衝液、0.1％トリトンX - 100。 </li></ol></li></ol> 2。架橋ソリューションの準備<ol><li>セルに適用する前に、すぐにDSPソリューションを準備。 DSPは、高度に疎水性であり、PBS /カルシウム/マグネシウムバッファに希釈する前に、DMSOに溶解してください。 DMSO 1mL中にDSPの40mgを溶解する。これは、DSP（DSPの分子量は404.42グラム/モルである）の100 mM溶液になります。 </li><li> ° C PBSにDSP / DMSOの希釈を促進するために、37〜PBS /カルシウム/マグネシウムの適切な量を温める。 各種プレートサイズに必要なボリューム： ウェルあたり6ウェルプレート2 mLの プレート当たり10cmの板を10mL プレート当たり15cmの板を20mL </li><li>温かいPBS /カルシウム/マグネシウムの各1 mLの場合、DSP / DMSOストック溶液の10μLを追加。すべてのDSPが溶解するまで繰り返す混合しながら降下することにより、DSP / DMSOストック溶液のドロップを追加。 PBS /カルシウム/マグネシウムの各1 mLに追加10μLのDMSOの制御ソリューションとなります。 </li><li> 10分よりもはや氷水浴中ですべてのPBS /カルシウム/マグネシウムソリューションを置きます。 </li></ol> 3。架橋のためにセルを準備します。 <ol><li>架橋や車両制御のインキュベーションのためのすべてのプレートに適合します氷水浴を準備する。 </li><li> 37℃のインキュベータからプレートを取り出し、氷水浴中にすぐにそれらを置きます。 </li><li>氷冷PBS /カルシウム/マグネシウムで細胞を2回洗浄する。あなたが（セクション2.2を参照）架橋剤インキュベーションに使用するのと同じボリュームを使用してください。 </li><li>回目の洗浄を削除し、車両制御やDSP架橋緩衝液のいずれかを追加。 </li><li> 2時間氷上でインキュベートする。あなたはすべてのセルがソリューションでカバーされていることを確認するには、約プレート20分ごとに確認する必要があります。あなたは、DSPの少量の溶液から沈殿することがあります。これは正常です。 </li></ol> 4。 DSPの反応の不活性化<ol><li> PBS /カルシウム/マグネシウム（PBS /カルシウム/マグネシウムの各1 mLの1 MトリスpH7.4の20μL）の20mMトリスpH7.4の1XのDSP焼入れ溶液を調製します。 </li><li>車両制御との架橋ソリューションを削除します。 </li><li>氷冷不活化ソリューションを追加し、15分間氷上でインキュベートする。 </li></ol> 5。細胞溶解<ol><li> DSPの消光のインキュベーションの間に、完全なプ​​ロテアーゼ阻害剤カクテルでバッファにトリトンX - 100 0.5％の細胞溶解バッファーを準備します。各1 mLの溶解バッファーのために完了の20μL50Xストック溶液を追加。 </li><li> 15分DSPの急冷インキュベーション後、PBS /カルシウム/マグネシウムで細胞を2回洗浄する。 </li><li>溶解バッファーを追加する+ 4で細胞や岩石への完全な℃で30分間。いくつかは、各種プレートのサイズについては、溶解バッファーの量を提案した。 <ul><li>ウェルあたり6ウェルプレートを0.5mL </li><li>プレート当たり10cmのプレートを1 mL </li><li>プレート当たり15cmのプレートを1 mL </li></ul></li><li>溶解後、プレートから細胞を収集するために、セルスクレーパーを使用してください。プロテアーゼ活性を最小限に抑えるために氷上でライセートを保つようにしてください</li><li> 4℃で15分間細胞ライセートをスピン·トップスピードで、ベンチトップのミニ遠心（15.000 × g）でのCを。 </li><li>新しいチューブに上清をピペットで。ペレットを捨てる。 </li><li>タンパク質レベルを分析し、免疫沈降に進んでください。 </li></ol> 6。免疫磁気降水ビーズの準備 注：この手順は、通常2時間の架橋インキュベーションを開始した後に直接起動される。 <ol><li> 、DYNAL免疫磁気ビーズを30μLを組み合わせるIPバッファー（1 ×緩衝液、0.1％トリトンX - 100）とscrewtopマイクロ遠心チューブに抗原特異的抗体の適切な量の500μL。ネガティブコントロールの抗体フリーと非特異的抗体管準備。抗体の適切な量は別々の実験中に個々の抗原に基づいて決定されるべきである。すべてのIPチューブにラベルを付けます。 </li><li> IPチューブを2時間室温でエンドオーバーエンドロッカーでインキュベートすることができます。また、架橋前に4 °日で一晩抗体でIPチューブをインキュベートする。 </li><li>インキュベーション後、チューブの底にビーズを収集するために迅速に10秒間のミニ遠心分離を行います。 </li><li>離れてチューブの底から磁気ビーズを引っ張ってくる磁気ホルダー、中にチューブをスライドさせて。安全に道のビーズを使用すると、簡単にすべての非結合抗体を洗い流すことができる。 </li><li>このようなゲルローディングチップ、またはインキュベーションバッファーと未結合の抗体を除去するためにP200チップとして小口径アスピレーターチップを使用してください。インキュベーションのボリュームが削除されるとすぐにIPバッファー1 mLに加える。 </li><li>静かに再サスペンドビーズ、チューブにフタをし、エンドオーバーエンド回転で5分間、室温でのすべてのIPチューブをインキュベートする。 </li><li> IPバッファの新鮮ミリリットルで一回より5分の洗浄ステップを（6.6を介してステップ6.3）繰り返します。 </li><li>架橋ライセートがビーズに追加する準備ができるまでIPチューブは最後の洗浄のインキュベーションに残すことができます。 </li></ol> 7。免疫磁気ビーズとの架橋ライセートをインキュベート<ol><li>最後の洗浄のスピン免疫磁気ビーズの後、ダウンして10秒間のミニ遠心インチその後、磁気ホルダーにチューブをスリップに進んでください。 </li><li>再び、小口径の吸引チップを使用して、チューブからインキュベーションのボリュームを削除します。すぐに免疫磁気ビーズを含むチューブに架橋した細胞溶解液の500μL（1μg/ mlで溶解液を仮定して）追加します。あなたがネガティブコントロールとしてペプチド競争を使用する場合、ペプチドの適切な量は、この時点で含まれるべきである。以前の実験は、適切なペプチドの競争条件を決定するために実行されるべきです。代わりに、ネガティブコントロールとしてライセート無料免疫磁気ビーズを持たせることができます。この場合は、すぐに免疫磁気ビーズへ+完全なライセートバッファー（1 ×バッファー+ 0.5％トリトンX - 100）の500μLを加える。 </li><li>優しくビーズを再懸濁します。再懸濁したビーズで2時間転倒回転で4℃でインキュベートしてください。 </li></ol> 8。ビーズからバインドされていない可溶化物を洗う<ol><li>かつてライセートが十分なインキュベーション時間を持っていた、ミニ遠心機で素早く10秒間のスピンを行います。その後、磁気ホルダーにチューブをスライドさせます。マグネットホルダーは、実験の残りを氷浴中で維持する必要があります。 </li><li>小口径アスピレーターチップを使用して、すべてのバインドされていないライセートを削除します。すべてのバインドされていないライセートが削除された直後にIPバッファー1 mLを加える。 </li><li>チューブにフタをし、静かにビーズを懸濁します。 </li><li>一度ビーズをビーズペレット化のステップ（8.1）を繰り返して再懸濁する。 </li><li>再びインキュベーションのボリュームのすべてをオフに吸引し、すぐにIPバッファー1 mLを加え、その後チューブをキャップ。 </li><li>優しくIPバッファーでビーズを再懸濁します。エンドオーバーエンドロッキングと4℃で5分間再懸濁したビーズをインキュベートする。 </li><li>ビーズの洗浄工程を（8.6経由で8.4）4回以上繰り返します。 </li></ol> 9。サンプルを変性し、ビーズから収集<ol><li>に免疫磁気降水管のすべてが洗浄された後、再堆積物ビーズ（8.1）とスライド管磁気ホルダー。 </li><li>免疫磁気ビーズに結合したタンパク質は変性条件下で除去される。最後のIPバッファの洗浄をオフに吸引し、磁気ホルダーからIPチューブを取り外します。 </li><li>再びチューブの下部にビーズプールにちょうどビーズペレット上にIPチューブに1 ×ゲルサンプルバッファーを追加します。サンプルバッファーの量は、使用するゲルのウェルサイズによって異なります。あなたが穏やかにゲルサンプルバッファーで再懸ビーズのすべてを取得するにはチューブの底をタップする必要がある場合があります。 </li><li>各IPチューブ用変性プロセス（9.3経由で9.2）を繰り返します。 </li><li>ゲルサンプルバッファーで再懸濁し、免疫磁気ビーズは、その後75℃に加熱されています°変性プロセスを完了するまで5分間。 </li><li>一度サンプルが変性されているそれは、ウェスタンブロッティングのためにSDS - PAGEゲル上で実行することができます。空に免疫磁気ビーズを遠心分離により、再ペレット化し、磁気ホルダー付きサンプルから削除することができます。 </li></ol> 10。抗原ペプチド（免疫アフィニティークロマトグラフィー）による架橋錯体の溶出。 <ol><li>免疫磁気免疫沈降管のすべての後にビーズ（8.1）とスライド管は、磁気ホルダーに再沈殿、洗浄されています。上清を吸引除去し、タンパク質複合体のimmunoisolationのために用いられる抗体が認識する抗原ペプチドを添加した緩衝液の10μlを加える。あなたは、効率的な溶出のための10から200μMからの範囲の濃度をテストする必要があります。 </li><li> 4℃で2時間ビーズをインキュベート℃を</li><li>マグネチックスタンドでビーズを入れて、慎重に溶出した物質の10 mLを回復する。この上清を保存します。 </li><li>すぐにバッファーでビーズを洗浄し、洗浄して廃棄する。ビーズを保存します。 </li><li> 1Xの濃度に保存された上清とビーズにゲルサンプルバッファーを追加します。 ℃で5分間75℃これらのチューブをインキュベートします。 </li><li> SDS - PAGEによりビーズとペプチドの溶出液を分析する。溶出液は、両方のSDS - PAGEの染色タンパク質または免疫ブロット（図1B）によるIgGの自由であるべきです。 IgGの自由なこの材料は、質量分析に適しています。 </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="図1" /> 図1。 AP - 3相互作用するタンパク質複合体や膜タンパク質の分離。HEK293細胞（）またはPC12細胞は、（B）車両の制御の有無（DMSO、奇数レーン図1A）またはDSP（偶数レーン図1Aまたはすべてのどちらかで処理した図1Bのレーン）。図、明確に抽出物を単独でビーズ（図1A、レーン3-4）、トランスフェリン受容体抗体（図1A、レーン5-6）、またはAP - 3δ抗体（図1A、レーン70から10のいずれかでインキュベートし、図1B、レーン1-10）。免疫複合体はSDS - PAGEサンプルバッファー（図1A）で溶出された、単独でのバッファまたは（図1B、レーン1-2）、またはアミノ酸680 710に対応するδの抗原ペプチドの濃度の増加を補充してバッファ（図1B、レーン3-10）：;：人間δ-アダプチン（1923266 GI AAD03777 NCBI）の。このペプチドは、δ抗体を結合する。ペプチド溶出した後、上清（S）とビーズ（B）は、免疫ブロット（図1B）によって分析した。 ;だけでなく、クラスリン重鎖（CHC）、BLOC - 1サブユニットのpallidin、、ホップ、複雑なサブユニットのvps33b：δ、β3、およびσ3サブユニットに対する抗体で検出されたAP - 3は、、以下の可溶性因子とcoprecipitates膜タンパク質のホスファチジルイノシトール4 - キナーゼII型α（PI4KIIα）と亜鉛トランスポーター3（ZnT3）。図中のペプチド溶出した上清中のIgGマウス重鎖が存在しないことに注意してください。 1B。

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSPは、12Åのスペーサーアームを持つ膜透過性、化学的に還元性架橋剤は、一時的なタンパク質相互作用1,2,3,4を安定させるために使用されます。ここでは、アダプタ複合体AP - 3 5をエンドソームから小胞への膜タンパク質を認識し、ソートする可溶性タンパク質複合体でこの戦略を例示する。 AP - 3は、選択的に亜鉛トランスポーターZnT3と脂質キナーゼ、ホスファチジルイノ?...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
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<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

携帯電話機械の動的な性質は、しばしば一過性および/または弱いタンパク質の関連付けに基づいて構築されています。これらの低親和性相互作用は、目的のタンパク質の周りにタンパク質ネットワークの分離と同定のための厳格な方法を排除する。化学架橋剤の使用は、精製のために生化学的な制限をバイパスして、不安定な相互作用の選択的安定化することができます。ここで私たちは12 Å、ジチオビス - （proprionateスクシンイミジル）（DSP）のスペーサーアームでホモ二官能性架橋剤を使用して培養中の細胞のための従順プロトコルを提示する。 DSPは、分子中に存在するジスルフィド結合の還元によって切断される。磁気ビーズによる目的タンパク質のイムノアフィニティークロマトグラフィーを組み合わせた架橋は、特に精製に耐えられないというタンパク質複合体の分離が可能になります。このプロトコルは、通常のウェスタンブロットの技法と互換性があり、それは質量分析法1によってタンパク質の同定のためにスケールアップすることができます。 ステファニーA. ZlaticとパールV.ライダーは、この作品にも同様に貢献した。

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

細胞透過性架橋剤DSP [ジチオビス - （スクシンイミジルプロピオネート）]安定一過性と不安定な相互作用<em&gt; in vivoで</em厳格なタンパク質複合体精製技術を用いてそれらの分離を可能にする&gt;、。ここでは、免疫沈降によるタンパク質複合体の分離に続いて培養で増殖架橋のセルのテクニックを紹介。

不安定な多タンパク質複合体の単離で in vivoで制御セルラー架橋及び免疫磁気アフィニティークロマトグラフィー

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

isolation-labile-multi-protein-complexes-vivo-controlled-cellular

Research

JoVE Journal

Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

17.2K ビュー.  Emory University. 細胞透過性架橋剤DSP [ジチオビス - （スクシンイミジルプロピオネート）]安定一過性と不安定な相互作用 in vivoで、。ここでは、免疫沈降によるタンパク質複合体の分離に続いて培養で増殖架橋のセルのテクニックを紹介。

ビデオ: Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

アミロイドペプチドに適用される変更されていないタンパク質（PICUP）の光誘起架橋

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

生物学

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

単一分子複合体の可視化インビボでの高度な蛍光顕微鏡を用いて

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME（免疫磁気電気化学的にリンクされた酵素）マイコトキシンの検出方法

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

高分子 - リガンド親和性を測定するための等温滴定カロリメトリー

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

細胞壁の弾性特性のAFMベースのマッピング：ティッシュ、携帯電話、および細胞内の解像度で

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...