我々は技術支援のためにZehavitフレンケルとサンドラTolchinskyに感謝。この作品に関連する研究は、イスラエル科学財団（1229年〜1207年）とドイツとイスラエルのプロジェクトの協力（DIP - DFG）からの補助金によってサポートされています。

以下のプロトコルは、全糖のか興味のある特定の糖タンパク質の糖鎖の解析を対象としています。手順は、プロトコル全体に述べたいくつかの変化と基本的にはどちらの場合も同じです。 <ol><li>各サンプルの90 mmディッシュ（サンプルは、異なるタイムポイントや治療法を表す可能性がある）で一晩増殖哺乳類細胞のサブコンカルチャを使用するN -結合型グリカンの代謝標識用のニーズ。このプロトコルは、NIH 3T3細胞に最適化されています。 </li><li> 10％透析FCSと5〜15分、4 mMのピルビン酸ナトリウムに付属している作りたての予め温めておいた（37℃）グルコースを含まない培地、5mlにインキュベートすることによりグルコースの細胞を飢えさせる。 </li><li> [ - 3 H 2]（65μCiの/ mlの低濃度は、総糖タンパク質の量のラベリングに使用される）マンノース標識、400μCiのを含む予め温めておいた（37℃）グルコースを含まない培地1mlで飢餓の媒体を交換してくださいと1時間の通常の組織培養条件で細胞をインキュベートする。 </li><li>チェイスに対応するサンプルがする必要がある一方、各サンプルから標識メディウムを外し、慎重に4℃の氷（これらのサンプルはパルスと呼ばれる）上にパルスと配置に対応するサンプルをPBS 2 mlを加え2mlで3回すすいだ予め温めておいた（37℃）、通常の完全培地、及びその後37℃CO 2インキュベーターに入れ° C、目的の追撃期間予め温めておいた定期的な培地5mlで。 </li><li>氷冷PBS 2mlでパルスのサンプル（以前氷上に置く）3回すすいでください。その後、細胞をセルスクレーパーを用いて2 mlのPBSでシャーレから掻き取り、そして2mlエッペンドルフチュー​​ブに配置されます。 </li><li> 16000Xg（6-10秒）で細胞を短期回転、上清を廃棄し、-80細胞ペレットを凍結℃に</li><li>チェイス時間の終了時追撃のサンプルについても手順5と6を実行します。 </li><li>冷凍庫からセルを削除し、バッファー300μlを添加することによって、それらを溶解させる、簡単にボルテックスして氷上で20分間インキュベートする、または、総糖タンパク質の分析の場合には、緩衝液Bで氷上でインキュベーション20分で、超音波処理が続く（4回、10秒、最大振幅）、その後5分間のサンプルを煮る。 </li><li> 4℃で20分間16000Xgでライセートを遠心新しいエッペンドルフチュー​​ブに上清を移し、ペレットを捨てる。 </li><li>合計糖タンパク質を分析する場合、分子ろ過と脱グリコシル化（ステップ17）に進みます。 H2Aの糖タンパク質の免疫沈降は、ここで紹介するため、（Repligen）プロテインA -アガロースビーズの20l/sample午前1時01分サスペンションを追加し、この例では、目的の糖タンパク質（私たちのウサギポリクローナル抗H2Aの抗体の3μL/サンプルアンチH2Aの抗体）、ステップ9から上清へ。 </li><li>常にゆっくりと回転して混合、4℃で4〜16時間のサンプルをインキュベートする。 </li><li> 30秒間16000Xgでビーズをスピンダウンし、注意深く上清を用いて真空を削除します。 </li><li> 500μlのバッファーDを追加し、ボルテックスで、ビーズを洗浄します。ステップ12のように回転し、上清を取り除く。洗浄を3回繰り返します。 </li><li>ビーズペレットに10μlの変性バッファー（NEBエンド- Hキットに付属）を追加し、5分（総蛋白質を分析する場合、脱グリコシル化は、マイクロコンフィルター、ステップ17で実行される）のサンプルを煮る。 </li><li> （30秒間16000Xg）ビーズをスピンダウンし、新しいエッペンドルフチュー​​ブに上清を移す。その後、遠藤Hの酵素の0.5μlを反応バッファー（また、NEB遠藤Hキットに付属）を10μlと一緒に各サンプルに追加され、サンプルは3時間、37℃でインキュベートする。 </li><li>タンパク質から放出される糖鎖を分離するために、試料を脱イオン水で5倍に希釈され、30kDaカットオフ、その後3分間14000Xg間遠心分離して、分子フィルター（マイクロコンUltracel YM30）の上に配置。脱イオン水と試料の繰り返しの遠心分離の50μLを適用する。このウォッシュアウトは、フロースルー保ち、さらに2回繰り返されます。 </li><li>あなたが全体の糖タンパク質を分析している場合、マイクロコンフィルターにライセートの上清を（ステップ9から）適用されます。 3分間14000Xgで繰り返し、遠心分離により3倍のバッファE、100μlの抽出液を洗浄してください。 10X遠藤Hの反応バッファーを3μlを追加し、エンドHの酵素1.5μlをマイクロコンの残留物に、そして37℃で3時間インキュベート℃にリリースグリカンは、ステップ16のように、3を繰り返し遠心分離による脱イオン水で溶出されています。 </li><li>必要に応じて、エンドH -抵抗性の糖タンパク質を含む手順16および17からretentatesは、​​その後37℃、緩衝液Cの15μlのN -グリコシダーゼFの200mUでインキュベートされる℃で16時間脱イオン水で溶出は、ステップ16のように行われます。 </li><li> Speed​​Vacコンセントレータで遠藤Hの反応フロースルー（ステップ16および17）を含むチューブを、と完全に試料を乾燥させ（これが45まで行うことができます℃で、このプロセスを加速する）。 </li><li> HPLC用サン​​プルを準備するには、HPLC溶媒（水、60:40、1％リン酸アセトニトリル）の12μl中、乾燥ペレットを懸濁します。 </li><li>定数の溶媒の流れ（1 ml /分）と圧力（1000と2000 psiの間に特定の値）にHPLCの装置を（フェリの列に接続されて）調整し、チューブを1分ごとに変更するにはフラクションコレクタを配置。 </li><li> HPLC装置にサンプルを読み込むと同時にフラクションコレクターを起動します。 </li><li>各サンプルの実行から、標準的​​なオリゴ糖混合物の実行から1 mlの48画分を集めた。 </li><li>各画分からのシンチレーションバイアルに500μlを、と水混和性のシンチレーション液3mlで内容を混ぜる。 </li><li>バイアルをロードし、シンチレーションカウンターにお読みください。 </li><li> CPMの読み出しは、分数の関数としてプロットされます。 </li><li>ピーク内のCPMの値は次のように、特定の糖鎖の種表す：分数を18-21 M8、M9のフラクション36から39にM5、フラクション22から26にM6、フラクション27から31にM7、フラクション32から35に対応フラクション40から42へG1M9とフラクション43から45へG2M9。 </li><li>各ピークの内画分のCPM値の合計は、特定の糖鎖の種の絶対量を反映している。より多くのマンノース残基を含む種がより多くのラベルを取得し、各糖鎖の種の&quot;相対的なモル量は、&quot;以下のように計算されているという事実を補正するために：各糖鎖の種の絶対量はそれに含まれるマンノース残基の数で割って。この値は、それぞれの特定の糖鎖の種のための&quot;合計のパーセントを&quot;得るために取得したすべての糖鎖生物種の相対的なモル量の総和で除算されます。 </li></ol><table border="1"><tbody><tr><td style="text-align:center;">チェース（H） </td><td style="text-align:center;"> 0 </td><td style="text-align:center;"> 4 </td></tr><tr><td>遠藤H（CPM）をリリースしました</td><td> 9万</td><td> 16800 </td></tr><tr><td> N -グリコシダーゼF（CPM）bでリリース</td><td> 20400 </td><td> 3900 </td></tr><tr><td>濃縮水（CPM）Cの糖タンパク質</td><td> 371700 </td><td> 127695 </td></tr><tr><td>濃縮水（CPM）、Dにおけるドリコール糖</td><td> 4350 </td><td> 540 </td></tr><tr><td>濃縮水のドリコール糖（％）E </td><td> 1.4 ± 0.7 </td><td> 0.2 ± 0.2 </td></tr></tbody></table> 表1。deglycosidasesあると糖タンパク質の試料におけるドリコール糖の存在のN -結合型糖鎖の放出の分析。 私たちは、NIH 3T3細胞からライセートにパルス - チェイスの手順を適用する。マイクロコンろ過した後、標識したN -結合型糖鎖は、パルスラベリング（高マンノース型）の後と複合型（ゴルジ処理された糖鎖）に対応するだろう追いかける期間後のN -グリコシダーゼFとのさらなる治療、と遠藤Hで主にリリースされました遠藤H抵抗性オリゴ糖。この定量分析はドリコール糖は、初期の残留物のラベルの微々たる割合を占めていると判断。 注意事項： <ol class="lalpha"><li> NIH 3T3細胞は、パルスが[2 - 3 H]で標識されたマンノースと完全培地で追いかけた。マイクロコンYM30を介して細胞を溶解し、ろ過した後、高マンノースN -結合型糖鎖は、エンドH.とのインキュベーションによりretentatesから解放された</li><li>からマイクロコンのretentates（）の遠藤H耐熱材料は、その後、シンチレーションカウンターで測定したN -グリコシダーゼFと放出オリゴ糖でインキュベートした。 </li><li>メタノール：：前駆体の糖脂質を除去する水午前10時10分03秒、およびunextracted材料を1N NaOHで可溶化し、シンチレーションカウンターでカウントエンドH処理の前に（）で得られたマイクロコンのretentatesは、​​クロロホルムで4回抽出した。 </li><li>ドリコール - 糖鎖は、（c）のように1に記載し、シンチレーションカウンターでカウントからの抽出物から精製した。 </li><li>残留液中の総CPMのドリコール糖のパーセントのための3つの実験の平均値（D）（C + D）。 </li></ol>代表的な結果<img src="/files/ftp_upload/1899/1899fig1.jpg" alt="図1" /> 図1。パルスチェイス未処理細胞のとプロテアソーム阻害時に合計NIH 3T3糖タンパク質の解析 1Hパルスラベリングの後で。[2 - 3 H]我々はいくつかglucosylatedと期待されるプロファイルを得前駆体は、（グルコース2マン9 N -アセチルグルコサミン2（G2M9）とG1M9）残りが、M9に存在するラベルとM8種のほとんどが、後者はすべてのグルコースのトリミング一マンノース残基（図1）の結果である。空きマンノースまたはその他の小規模な前駆体は、精製の徹底を証明する、検出されなかった。かなり長い8Hチェースに続いてそこにM7 - 6とM5の少量にトリミングをより広範であったが、主要な種はM9とM8（図1 B）が続きました。チェイスはプロテアソーム阻害剤（30μMのMG - 132）の存在下で行われた場合、これらの同じトリミング種のマイナー蓄積（図1 C）のみがあった。 <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="図2" /> 図2。合計糖タンパク質に比べてERAD基質の糖鎖の定量分析。 （A）図1に示すような実験では、各オリゴ糖の種の相対的なモル量は、マンノース含有量に基づいて算出した。私たちは、それぞれの糖鎖生物種で得られたCPMの値を変換し、すべての種の存在の相対的なモル量の総和に対する各種の％は、2つの実験の平均の追求を時間の関数としてプロットした。（B）と同様に（）ERAD基質ASGPR H2Aから放出その糖鎖を除いて分析した後、存在下で4時間追いかけるためにプロテアソーム阻害剤MG - 132の存在下または非存在下で4時間までのパルスラベルとチェイスは、。（C）の値からMG - 132の（A）と（B）ASGPR H2Aおよび糖タンパク質のプールのために比較されます。ER内のプロセスをトリミングN -結合型糖鎖の（D）スキーム。砂糖は、ゴルジ体以降に終了するものでM9 - 8と比較しERADを対象とするタンパク質（R）にリンクされているM6 - 5につながるプロセスをトリミング。結果は、ERADのプロセスが残っている5月6日マンノース残基を有する種を生成するためにN -結合型糖鎖のトリミングマンノースに関連付けられていることを示している。

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

パルスチェイス生細胞における糖鎖の分析HPLC分離とは、糖タンパク質の一生を通じてオリゴ糖の構造変化のダイナミクスを研究するための方法を提供する。このような変化がERのフォールディング、品質管理および売買システム2-5の信号を生成に関与していることを成長証拠がある。方法はないだけ関心のある特定の糖タンパク質のためだけでなく、我々は対照的な、特定のERADの基?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>600E Multisolvent delivery System controller</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT062710</td>
<td>&nbsp;</td>
<tr>
<td>Acetonitrile LiChrosolv (gradient grade for liquid chromatography)</td>
<td>&nbsp;</td>
<td>Merck Bioscience</td>
<td>1.0003</td>
<td>&nbsp;</td>
<tr>
<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC503024 or 4208</td>
<td>&nbsp;</td>
<tr>
<td>Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5301 000.016</td>
<td>&nbsp;</td>
<tr>
<td>Dialyzed Foetal Calf Serum</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-011-1A</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen Cell Culture</td>
<td>41965039</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modi&#xFB01;ed Eagle's Medium - glucose free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5030</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>EDTA disodium salt (Titriplex Iii)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1084180250</td>
<td>&nbsp;</td>
<tr>
<td>Endo Hf Kit (1,000,000 units/ml)</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>p0703S</td>
<td>&nbsp;</td>
<tr>
<td>Foetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-001-1A</td>
<td>&nbsp;</td>
<tr>
<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
<tr>
<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
<tr>
<td>Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol)</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>NET570A</td>
<td>&nbsp;</td>
<tr>
<td>N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>474790</td>
<td>&nbsp;</td>
<tr>
<td>N-glycosidase F (1000 units/ml)</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11365177</td>
<td>&nbsp;</td>
<tr>
<td>N-octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>O3757</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 cells</td>
<td>&nbsp;</td>
<td>American Type Culture Collection (ATCC)</td>
<td>CRL-1658</td>
<td>&nbsp;</td>
<tr>
<td>Opti-Flour</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>6013199</td>
<td>&nbsp;</td>
<tr>
<td>Phosphoric acid solution ( 49-51%, for HPLC)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>79607</td>
<td>&nbsp;</td>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2714</td>
<td>&nbsp;</td>
<tr>
<td>Protein A-Sepharose beads</td>
<td>&nbsp;</td>
<td>Repligen</td>
<td>IPA300</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>30970</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Bio-RAD</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>342483</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution (100mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Spherisorb NH2 Column, 5 &mu;m, 4.6 x 250 mm</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>PSS831115</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>BDH</td>
<td>306324N</td>
<td>&nbsp;</td>
<tr>
<td>Vibra-Cell ultrasonic processors VCX 750</td>
<td>&nbsp;</td>
<td>Sonics and Materials, Inc.</td>
<td>690-003</td>
<td>&nbsp;</td>
<tr>
<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 stably expressing H2a 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Glcの添付ファイル<sub&gt; 3</sub&gt;マン<sub&gt; 9</sub&gt; N -アセチルグルコサミン<sub&gt; 2</subERでの新生ポリペプチドに対するオリゴ糖&gt;前駆体は分泌蛋白質に共通の変更です。この変更は、いくつかの生物学的プロセスに関与されたが、その機能の追加の側面は、糖タンパク質の品質管理や人身売買のシグナルの生産におけるその役割の最近の証拠で、浮上している。従って、N -結合型糖鎖とその処理に関連する現象が集中的に研究されている。最近プロテオーム解析のために開発されてきた方法は、大幅に糖タンパク質のN -結合型グリカンの特性を改善している。それにもかかわらず、彼らは関与する糖鎖プロセッシングの動態への洞察を提供していません。このために、ラベリングやパルスチェイス解析プロトコルは通常、複雑であり、非常に低い収率を与えることに使用されます。ここでは、代謝的に標識したN -結合型糖鎖の単離と解析のためのシンプルな方法を説明します。プロトコルは[2細胞のラベリングに基づいている -<sup&gt; 3</sup&gt; H関心のどちら特に免疫沈降したタンパク質からまたは分子濾過（アミコン）に続いて遠藤HとN -グリコシダーゼFと順次治療による一般的な糖タンパク質プールから]マンノース、変性溶解し、オリゴ糖の酵素的放出。この方法では分離されたオリゴ糖は単糖類の分解能で、それらを含有する単糖単位の数に応じて様々な糖鎖構造の間に差別を可能にするHPLC分析のための入力として機能します。この方法を用いて、我々は通常の状態にし、プロテアソーム阻害下でパルスチェイス後の全細胞の糖タンパク質の高マンノースN -結合型糖鎖のプロファイルを研究することができた。これらのプロファイルは、免疫沈降した小胞体関連分解（ERAD）基板から得られたものと比較した。我々の結果は、ほとんどのNIH 3T3細胞の糖タンパク質は比較的安定していることと、それらのオリゴ糖の大部分が男にトリミングされていることを示唆している<sub&gt; 9月8日</sub&gt; N -アセチルグルコサミン<sub&gt; 2</sub&gt;。対照的に、不安定なERADの基質は、人間にトリミングされています<sub&gt; 6月5日</sub&gt; N -アセチルグルコサミン<sub&gt; 2</subこれらの種をもつ&gt;と糖タンパク質は、プロテアソームによる分解の阻害により蓄積する。

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

我々は、哺乳類細胞におけるそれらの生合成後の糖タンパク質の初期の生活を通じて、N -結合型グリカンの変化の分析のための方法を説明します。これは、代謝的に標識した糖鎖、糖タンパク質およびHPLCによる検討からの酵素遊離のパルス - チェイス分析によって達成されます。

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

Research

JoVE Journal

Biology

15.1K ビュー.  Tel Aviv University. 我々は、哺乳類細胞におけるそれらの生合成後の糖タンパク質の初期の生活を通じて、N -結合型グリカンの変化の分析のための方法を説明します。これは、代謝的に標識した糖鎖、糖タンパク質およびHPLCによる検討からの酵素遊離のパルス - チェイス分析によって達成されます。

ビデオ: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

生物学

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

哺乳動物細胞におけるDNA二重鎖切断（DSB）修復の解析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

哺乳動物細胞におけるRNAiスクリーニングのためのミッションesiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

マイクロインジェクションによる哺乳動物細胞におけるmRNA核外輸送の速度論の解析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳類のリンカーヒストンサブタイプの発現解析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

哺乳動物細胞におけるヘム合成レベルの測定

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...