この作品は、公衆衛生のカリフォルニア部からラリーL. Hillblomアルツハイマー病協会から財団、IIRG - 07から58334まで、および07から65798からNIH / NIA、2005/2Eからの補助金AG027818とAG030709によってサポートされていました。

1。ペプチドの準備<ol><li>秤量〜というラベルの付いた、シリコンでコーティングされた、低吸着剤マイクロチューブに微量天秤および転送を使用して凍結乾燥したペプチド100〜200μgの。ここで、我々は、Aβ40のヒト配列、Aβ42、カルシトニン、およびGRFを使用してください。 </li><li>ここで、我々は均一な、集約フリーの準備を取得するためにペプチド1,1,1,3,3,3 - ヘキサフルオロ-2 - プロパノール（HFIP）で前処理を使用してください。予め形成された凝集体は、実験5の間で再現性の低下につながるアミロイド蛋白質の迅速な集約を、誘発するため、この手順が必要となります。そのようなろ過やSECのような他の方法もPICUP 6の集約-フリーの準備を取得するために使用することができます。 </li><li> HFIPを有するペプチドを治療するために、適切な保護（HFIPは揮発性と毒性がある）を身に着けているヒュームフード内部の氷の上にHFIPコンテナを事前に冷やします。 250mlのボトルの冷却は、通常、10〜15分が必要です。 0.5mMの名目上のペプチドの濃度を得るためにペプチドの凍結乾燥を含むあらかじめ冷却管にHFIPを追加。 </li><li>室温で5分間水浴の超音波処理のペプチドソリューションを超音波処理してください。 </li><li>穏やかに攪拌し、室温で30分間チューブをインキュベートする。 </li><li>氷上でチューブを（1分間）寒さ、および標識された0.6 mlの低吸着チューブ10 - 50 -μLアリコートにソリューションを分ける。 </li><li>窒素の気流下で蒸発させてHFIPを削除する、または一晩換気フードのオープンチューブを残す。一晩蒸発するため、ラック内の開いているチューブを配置し、ほこりや微粒子の汚染を防ぐために、キムワイプの大きなシートでそれらをカバーしています。 </li><li>凍結乾燥機で真空下での残りのHFIP、または30分間、または2時間真空入口に接続されているexsiccatorで遠心濃縮器をカラカラに乾燥させる最終製品は、マイクロチューブの下部にあるペプチドフィルムになります。適切に無水亜硫酸場合、チューブを-20または-80℃で長期間（数ヶ月）のために気密保存することができます。 </li></ol> 2。 HFIP処理ペプチドを可溶化すると写真が架橋<ol><li>架橋反応は、架橋と消光試薬を準備する一つのニーズにHFIP処理ペプチドを可溶化する前に。 </li><li>過硫酸アンモニウム（APS、ミスター228.2グラム/モル）を秤量し、10mMリン酸ナトリウム、20 mM溶液、pH7.4を準備する。溶液が透明になるまでボルテックスを用いて混ぜる。 </li><li> 10 mMリン酸ナトリウム中のトリス（2,2 - ビピリジル）ジクロロ（II）六水和物（RuBpy、氏748.63グラム/モル）、pH7.4の1mMの溶液を調製します。ボルテックスを用いて混合し、完全な溶解を確認してください。アルミ箔を使用して光からチューブを保護します。 </li><li>架橋結合は、次のSDS - PAGE解析のために、便利な消光試薬は、2の5％β-メルカプトエタノール× SDS - PAGEサンプルバッファーです。また、脱イオン水または適当なバッファー中で1 Mジチオスレイトール（DTT、ミスター154.5グラム/モル）を使用することができます。 </li><li> HFIP処理ペプチド膜を希NaOHで最初に溶解され、その後、リン酸ナトリウム緩衝液は、〜30μMのペプチドの濃度を得るために追加されます。追加60mMのNaOHは水酸化ナトリウムと水がそれぞれ10と、最終的な体積の45％を、構成するようなペプチドフィルムを含むチューブに精製水が続きます。先端、ピペッティングにより混合し、水浴の超音波処理で5分間超音波処理を用いてマイクロチューブの内壁からペプチドフィルムをこすり落とす。 </li><li> 45パーセント20 mMリン酸ナトリウム、pH7.4の、ピペッティングにより混和し、10分間16,000 gで遠心分離を追加。ネガティブコントロールの消光試薬と混合交差を解くにリンクされたペプチドのアリコートを脇に置きます。 </li><li> 1秒間にカメラのシャッターの遅延を調整する高い照射時間で、豊富なラジカル反応は、タンパク質分解を引き起こす可能性があります。カメラのシャッターをロードする。照射時間は、新しいタンパク質を持つメソッドを使用するときに最適化する必要があります。 </li><li>典型的なPICUP反応は20μlの反応容量で実行されます。 、薄肉、明確な0.2 mlのPCRチューブにペプチド溶液18μlを。 </li><li>ペプチド溶液に、1μlのAPSが続く1μlのRuBpyを追加し、（反応混合物中のRuBpyとAPSの最終濃度はそれぞれ0.05および1mMの、となる）ピペッティングにより試薬を混合する。 </li><li>すぐに1.8 mLのガラスバイアルの内部に反応チューブを置きます。カメラ本体の前面に取り付けられたベローズ内部にバイアルを置きます。レンズプロテクターを取り付け、試料はベローズ内部の1秒間照射されるように、シャッターを押してください。 </li><li>サンプルの照射後、すぐにバイアルからベローズとPCRの管からバイアルを取り出す。すぐに1μlのDTTまたはPAGEサンプルバッファーを減らす10μlを添加することにより反応をクエンチ。各ペプチドのアリコートに対して反応を繰り返します。 </li><li>反応混合物は、現在7日間より長くのためのストレージのために-20℃で凍結またはSDS - PAGEおよび銀染色によって分析する前に氷上で保存することができます。 </li></ol> 3。 SDS - PAGEとスライバー染色架橋ペプチド製品の<ol><li>ルーチンSDS - PAGEおよび銀染色、架橋ペプチドを可視化するために実行されます。 </li><li>レーンあたりペプチドの約130 pmolのを取得するために車線ごとの反応混合物の5μlをロードします。 </li><li>比較のために交差を解くにリンクされたペプチドの類似の額が含まれております。ペプチドのバンドの分子量を視覚的に近似するための標準的なタンパク質ラダーをも含まれています。 </li><li>標準的なゲル電気泳動装置を用いてゲルを実行します。我々は、InvitrogenからX細胞SureLockミニセルシステムを使用し、Invitrogen社出版IM - 1002、NOVEXプレキャストゲル電気泳動のガイドにしたがって銀染色を行う。 </li></ol>パート4：代表的な結果（図1） <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="図1" /> 図1：交差を解くにリンクを示す銀染色ポリアクリルアミドゲル（ - ）と（+）光架橋GRF、カルシトニン、Aβ40、Aβ42および SDS - PAGEとPICUP生成されたAβ40オリゴマーの銀染色分析は、より高いオリゴマー7の存在量の急激な減少が続く四量体を介して単量体の約類似のバンド強度を、示す。交差を解く連動型Aβ40は、モノマー7のそれと一致して氏に移行されます。 Aβ42は異なるオリゴマーのサイズ分布を示しています。 Aβ42オリゴマーは2-3グループ8を備える。最初のグループ、三量体を介して単量体は、増加するオリゴマーの順で減少する強度を表示します。第二のグループでは、ガウス状の分布は、五量体および六量体で最大で、四量体と量体との間で観察される。ここに示されていない第三のグループは、、氏〜30,000-60,000ダ8のオリゴマーが含まれています。これらの高いMRのオリゴマーは、一般的にSEC -孤立LMWAβ42がろ過またはHFIPの治療6で準備ができていないペプチドを用いて観察される。交差を解くにリンクされたAβ42は、主に二つのバンド、単量体のバンドとSDS 5,9により誘導される人工物である幅広いトリマー/四量体のバンドを、生成する。カルシトニンは強くダイマー、トリマー、テトラマーおよび7のプレ存在を示唆し、四量体を介して地域のモノマーの単調指数関数的に減少から分岐オリゴマーのサイズ分布が得られます。コントロールペプチド、GRFは、二量体からオリゴマーの見かけの相対量が増加して分子量を指数関数的に減少するような六量体までの範囲で単調性オリゴマーの分布を生成します。他の実験では、より高いオリゴマーは、7を視覚化することができます。正確な数とオリゴマーのバンドの相対強度は、実際のタンパク質濃度、ゲル上にロードされたタンパク質の量、および銀染色のプロトコルの開発のステップのために使用される時間に応じて実験の間で多少異なる場合があります。

<ol>
	<li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. USA. 96, 6020-6024 (1999).</li><li>Kluger, R., Alagic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking+and+protein-protein+interactions&#8212;a+review+with+illustrative+protocols.">Chemical cross-linking and protein-protein interactions&#8212;a review with illustrative protocols.</a> Bioorg. Chem. 32, 451-472 (2004).</li><li>Tomohiro, T., Hashimoto, M., Hatanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+chemistry+and+biology:+development+of+multifunctional+photoaffinity+probes.">Cross-linking chemistry and biology: development of multifunctional photoaffinity probes.</a> Chem. Rec. 5, 385-395 (2005).</li><li>Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+protein+oligomers&#8212;what+you+see+is+not+always+what+you+get.">Neurotoxic protein oligomers&#8212;what you see is not always what you get.</a> Amyloid. 12, 88-95 (2005).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+aggregate-free,+low+molecular+weight+amyloid-&#946;+for+assembly+and+toxicity+assays.">Preparation of aggregate-free, low molecular weight amyloid-&#946; for assembly and toxicity assays.</a> Methods Mol. Biol. 299, 3-9 (2005).</li><li>Bitan, G., Lomakin, A., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. D., Breese, R., Dodson, E. C., Acton, P., Keller, P. M., Yeager, M., Wang, H., Shughrue, P., Kinney, G., Joyce, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+state+characterization+of+amyloid+&#946;-derived+diffusible+ligands.">Solution state characterization of amyloid &#946;-derived diffusible ligands.</a> Biochemistry (Mosc). 45, 15157-15167 (2006).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+photochemical+cross-linking&#8212;a+new+tool+for+studies+of+metastable,+amyloidogenic+protein+assemblies).">Rapid photochemical cross-linking&#8212;a new tool for studies of metastable, amyloidogenic protein assemblies).</a> Acc. Chem. Res. 37, 357-364 (2004).</li><li>Piening, N., Weber, P., Hogen, T., Beekes, M., Kretzschmar, H., Giese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+crosslinking+of+prion+protein+oligomers+and+prions.">Photo-induced crosslinking of prion protein oligomers and prions.</a> Amyloid. 13, 67-77 (2006).</li><li>Li, H. T., Lin, X. J., Xie, Y. Y., Hu, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+events+of+&#945;-synuclein+oligomerization+revealed+by+photo-induced+cross-linking.">The early events of &#945;-synuclein oligomerization revealed by photo-induced cross-linking.</a> Protein Pept. Lett. 13, 385-390 (2006).</li><li>Vollers, S. S., Teplow, D. B., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Peptide+oligomerization+state+using+rapid+photochemical+crosslinking.">Determination of Peptide oligomerization state using rapid photochemical crosslinking.</a> Methods Mol. Biol. 299, 11-18 (2005).</li></ol>

PICUPは、安定したタンパク質複合体2を研究するためにもともと開発されました。メソッドは、Aβ10、プリオンと疾患関連PrP Scが11、およびα-シヌクレイン12を含む準安定アミロイドタンパク質の集合体の定量的研究、に後から適用されました。 PICUPの実験を設計する際に考慮しなければならない最も重要な要因は、試薬の化学量論、照射時間、及び試料調製の手順で?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

毒性オリゴマーへのアミロイド蛋白質のアセンブリは、アルツハイマー病、パーキンソン病、およびハンチントン病、遺伝性の筋萎縮性側索硬化症、および2型糖尿病を含むタンパク質のミスフォールディング病の病因に重要な出来事です。これらのタンパク質のアセンブリの準安定な性質のおかげで、それはそのような電気泳動、クロマトグラフィー、蛍光、または動的光散乱などの古典的な方法を用いて定量的にそのオリゴマーのサイズ分布を評価するのは困難である。アミロイド蛋白質のオリゴマーはオリゴマーがモノマーに解離すると同時に、大きなアセンブリに関連付けるれる準安定混合物として存在している。 PICUPは共有結合架橋やドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（SDS - PAGE）やサイズ排除クロマトグラフィ（SEC）などの分画法と組み合わせた場合、PICUPがクロスする前に存在したオリゴマーのサイズ分布のスナップショットを提供することによりオリゴマーの個体数を安定化 - リンク。したがって、PICUPは準安定タンパク質集団の可視化と定量分析を可能にし、シーケンスの変更とオリゴマー化の間に組み立てと解読の関係を監視するために使用することができます。<sup&gt; 1</sup&gt;。機構的に、PICUPは、Ruの光酸化を含む<sup&gt; 2 +</sup&gt;トリス（ビピリジル）ルテニウム（II）錯体（RuBpy）のRuに<sup&gt; 3 +</sup電子受容体の存在下で可視光照射による&gt;。 RU<sup&gt; 3 +</sup&gt;ラジカルタンパク質を生成し、隣接するタンパク質の分子から電子を抽象化することのできる強力な一電子酸化剤です。<sup&gt; 1,2</sup&gt;。ラジカルは不安定な、非常に反応性種であり、したがって、内と分子間反応のさまざまな急速に消える。ラジカルは、その後、水素原子を失い、安定した、共有結合している二量体を形成する二量体のラジカルを形成する別のタンパク質の単量体と反応する不対電子の高エネルギーを利用することができる。二量体は、次に高次のオリゴマーを形成する単量体または他の二量体と同様の機構を介してさらに反応する可能性があります。その他の写真または化学的方法で架橋相対PICUPの利点<sup&gt; 3,4</sup&gt;短い（≤1秒）の非破壊可視光への曝露、不要に含まれています<i&gt;事前に事実上の</i&gt;ネイティブ配列​​の改変、および長さゼロの共有結合架橋。さらに、PICUPは生理的パラメータを含む、広いpHおよび温度範囲内のタンパク質の架橋を可能にします。ここで、我々は3つのアミロイド蛋白質40の架橋にPICUPのアプリケーションを示す - と42残基のアミロイドβ-タンパク質変異体（Aβ40およびAβ42）、及びカルシトニン、および制御のタンパク質、成長ホルモン放出因子（GRF）。

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

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

変更されていないタンパク質（PICUP）の光誘起架橋は、準安定タンパク質混合物のオリゴマーのサイズ分布の特性評価が可能になります。とアミロイドβ-蛋白質、およびカルシトニンの42残基のフォーム、およびコントロールペプチド成長ホルモン放出因子 - 私たちは、3つの代表的なアミロイドペプチド40にPICUPのアプリケーションを示しています。

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, ...

photo-induced-cross-linking-unmodified-proteins-picup-applied-to

Research

JoVE Journal

Biology

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

21.2K ビュー.  University of California, Los Angeles. 変更されていないタンパク質（PICUP）の光誘起架橋は、準安定タンパク質混合物のオリゴマーのサイズ分布の特性評価が可能になります。とアミロイドβ-蛋白質、およびカルシトニンの42残基のフォーム、およびコントロールペプチド成長ホルモン放出因子 - 私たちは、3つの代表的なアミロイドペプチド40にPICUPのアプリケーションを示しています。

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

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

タンパク質の電気泳動分離

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

生物学

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

ゲルでタンパク質を染色

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

複雑な生体試料から選択的グリコシル化ペプチドを豊かにするためにレクチンHPLC法

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

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

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

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

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

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 ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

から細胞のサブセットの遺伝子発現プロファイリングへの応用レーザーマイクロダイセクションショウジョウバエウィングディスク

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

で産生された組換えジスルフィド豊富な毒タンパク質に印加される高スループット定量的発現スクリーニングおよび精製<em&gt; E.大腸菌の</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

からのペプチドおよびタンパク質との組換えエラスチン様ポリペプチドおよびその融合の非クロマトグラフィー精製<em&gt;エシェリヒア·コリ</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF質量分析およびカスタムデータベースを使用すると、ユニークな洞窟環境（カーチェナー洞窟、AZ、USA）を常在菌を特徴づけるために

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

比色金ナノ粒子アッセイに適用される構造スイッチングアプタマーを選択するための方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

質量分析のためのペプチドに組織および消化から細胞外マトリックスタンパク質の濃縮

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...