この研究は、RWへの退役軍人の米国エネルギー省、およびNIHのグラントR01CA138528から優秀賞によってサポートされていました。

発現構築物の調製<ol><li>プロトコルを開始する前に、N末端エピトープタグ（MycのまたはHA）、および細菌細胞から高レベルのタンパク質発現および精製のための基質インフレームグルタチオン-S-トランスフェラーゼ（GST）とを有する哺乳動物発現ベクターにクローンPRMTs標準的な分子生物学的技術を用いて、溶解物、。 </li></ol> PRMTs 2。精製<ol><li>ヒト気管支上皮細胞（Beas2B）を維持するために、100mmの培養皿を使用してください。継代細胞3日ごとにし、それらがコンフルエンスまで成長させてください。 </li><li>トランスフェクションの前日に、100mm培養皿中の10mlの培地中7.5×10 5の細胞を播種する。スワールは細胞を均一に分散し、5％加湿CO 2インキュベーター中、37℃でインキュベートする。 </li><li>翌日、培地を吸引除去し、血清および抗生物質を含まない培地の5ミリリットルを追加します。旋回やメディアを吸引除去する。このステップを繰り返し1最終的にはより多くの時間、および血清/抗生物質を含まない培地の5ミリリットルを追加し、トランスフェクションに進んでください。 </li><li>無菌1.5ミリリットルのプラスチック遠心管中の無血清培地を750μlにプラスミドDNA6μgの希釈する。 </li><li>別の1.5ミリリットルのプラスチック遠心管では、無血清培地750μlの中にトランスフェクション試薬30μlの希釈する。 </li><li>希釈したDNA（ステップ2.4​​）を含有するチューブに希釈したトランスフェクション試薬溶液（ステップ2.5）を転送します。数回ピペッティングにより穏やかに混合します。 </li><li> 15分間室温でトランスフェクション混合物をインキュベートする。 </li><li> 15分後、細胞の上に静かにトランスフェクション混合物をピペットで、スワールが均一に混合し、5％加湿CO 2インキュベーター中、37℃でインキュベートする。 </li><li> 3時間後、細胞からトランスフェクション混合物を含有する培地を吸引する。血清および抗生物質を補充した新鮮な細胞培養培地を加える。 </li><li> 5％加湿CO 2中、37℃でインキュベートする48時間インキュベーター。 </li><li> 48時間後、溶解緩衝液1ml中に細胞を溶解し（1×リン酸緩衝生理食塩水、1％のNonidet P-40、0.5％デオキシコール酸ナトリウム、0.1％ドデシル硫酸ナトリウム、および1μg/ mlのphenlymethylスルホニルフルオリド）。セルリフターのプラスチック遠心管に溶解物を収集し、氷上で30分間溶解物をインキュベートする。 </li><li>細胞破片からの溶解物をクリアするためにベンチトップ遠心機で10分間15,000×gで溶解物を遠心。 [*これは、高度にメチル化アッセイのための酵素の精製に進む前に、抗HA抗体を用いた溶解物のアリコートの免疫ブロッティングを経由して、HA-PRMT1の発現を確認することをお勧めします。] </li><li> PRMTsの精製のために、リン酸緩衝生理食塩水（PBS）中の抗HAアフィニティマトリックスの50:50懸濁液の30μlを用いた細胞溶解物3mgのインキュベートし、チューブローテーター中で4℃で一晩回転させる。 </li><li>翌日、収集するために2分間ベンチトップ遠心分離機で14000×gでチューブを回転ビーズ。上清を破棄し、2、14,000×gで遠心分離し、各洗浄で[、20mMトリス（pH8.0）に150mMのNaCl、5mMのEDTA及び1％トリトン-X-100]をビーズはRIPA 1mlのバッファーで3回洗浄分。 3回目の洗浄の後、メチル化緩衝液100μlで一回ビーズを洗浄緩衝液（50mM Tris pH8.5の、20のKCl、10mMのMgCl 2、1mMのβ-メルカプトエタノール、100mMのスクロース）。遠心分離後、上清を除去します。固定化PRMTsを含むビーズは、メチル化アッセイで使用される準備ができている。 [*これは非常にすぐにメチル化アッセイに固定化PRMTsを使用することを推奨します。] </li></ol>基質の精製<ol><li> GST-G3BP1の単一コロニーを接種し、100μg/ mlのアンピシリンを含むルリア - ベルターニ（LB）培地5mlにBL21を形質転換した。 250rpmで振盪しながら37℃で一晩培養を育てる。 </li><li>翌日、新鮮なLB培地と続き、50mlの一晩培養物を移す（これは、約2時間かかります）0.5〜0.7のOD 600まで成長するinue。 </li><li> OD 600が 0.5〜0.7に達したときに、1 mMの最終濃度になるようにイソプロピル-β-D-チオガラクトシド（IPTG）を添加し、250rpmで振盪しながら37℃でさらに4時間培養を続ける。 </li><li> 10分間1500×gでの遠心分離によって細胞を採取し、-80℃でGST精製またはストアのペレットを処理する。 </li><li>再懸濁溶解緩衝液5ml（プロテアーゼ阻害剤、リゾチーム100μg/ mlの、及び0.1％のTriton-X-100と1×PBS）における細菌ペレットを、30分間、37℃でライセートをインキュベートする。この時、溶解物は、細菌ゲノムDNAのリリースに濁った、粘性が表示されます。 </li><li>超音波処理（30％の振幅に1秒の10パルスごと）によりゲノムDNAを剪断。 </li><li> 10分間15000×gでの遠心分離によって溶解物をクリアし、15mlチューブに透明な上清を移す。上清をcentrifuを繰り返し明確ではない場合ゲーションプロセスをもう一度。 </li><li>一方、氷冷PBSで2回、GST-セファロースビーズを洗浄し、PBS中のGST-セファロースの50:50懸濁液を作製。クリア細菌細胞溶解液に、GST-セファロース懸濁液200μlを加え、1時間、4℃で回転させます。 </li><li> 1時間後、ビーズを収集するために3分間500×gでチューブを遠心する。ビーズは氷冷PBSで3回洗浄する。 </li><li>最後の洗浄後、溶出緩衝液100μl（50mMトリスpH8.0で、150mMのNaCl、30mMのグルタチオン）中でビーズを再懸濁し、10分間4℃で回転させる。 </li><li> 2分間15000×gでチューブを遠心し、別のプラスチック製遠心管に精製したタンパク質を含む上清を移す。精製タンパク質の濃度を推定するためにウシ血清アルブミン（BSA）の既知量と一緒に精製されたタンパク質を5μlにSDSポリアクリルアミドゲル電気泳動（SDS-PAGE）分析を行う。 </li></ol> 4。メチル化アッセイ注：放射性同位元素の仕事のために、放射性同位体で実験を行う上で機関のプロトコルに準拠して指定された別の領域に、次の手順を実行します。 <ol><li>精製されたGST-基質および1×メチル化バッファー（50mMトリスpH8.5の、20のKCl、10mMのMgCl 2、1mMのβ-メルカプトエタノール、100mMのスクロース）、1μCiの含有するマスターミックス2μgの追加のS-アデノシルL-[メチル- 3 H]メチオニン、水、固定化PRMTs（ステップ2.13）に10μlの最終体積にし、1時間、30℃で反応をインキュベートする。 </li><li> 1時間後、2×SDS試料緩衝液10μlを添加することにより反応を停止。 10分間95℃での試料の変性後、SDS-PAGEゲルでサンプルを分離する。 </li><li>以降、半乾燥電気泳動トランスファーを用いてニトロセルロース膜にSDSゲルから分離されたタンパク質を移す。 </li><li>転写後、を有する膜をカバーutofluor（オートラジオグラフィーイメージインテンシファイア）と、穏やかに室温で2時間揺らし。 2時間後、オートフルオーを捨てる、後で使用するために保持します。 [*放射性物質などのチューブにラベルを付ける] </li><li>迅速空気は、ろ紙上の膜を乾燥ビニール袋に入れて密封し、-20℃でX線フィルムに膜を露出させる。十分な露光時間は5〜30日の間の範囲である。 </li></ol> PRMT発現と基質の等しい負荷の5。確認<ol><li>十分なエクスポージャーを達成した後、ブロッキングバッファーを含むプラスチック箱[トゥイーン20（TBST + 3％脱脂乳）をトリス緩衝生理食塩水]を、膜を転送し、穏やかに1時間揺らし。 </li><li>放射性廃棄物としてブロッキングバッファーを捨てます。 4℃で一晩TBST中：（1,000希釈1）抗HA抗体で膜をインキュベート 穏やかに揺らしながら°C、。 </li><li> TBSTで3回で膜を洗浄し、HRP標識抗マウス二次抗体で膜をインキュベート穏やかに揺らしながら1時間室温でTBST中：（1 5000希釈）。 </li><li> 1時間後、TBST 3倍の膜を洗浄し、HRP基質を有するタンパク質の化学発光検出を行う。 </li><li> HAタグ付きPRMTsを検出した後、室温で5分間Ponseau-S染色液で膜をインキュベートする。すぐにGSTタグ基質を検出するために、水と、膜3回洗う。画像をスキャンします。 </li></ol>

<ol>
	<li>Paik, W. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+methylase+I.+Purification+and+properties+of+the+enzyme.">Protein methylase I. Purification and properties of the enzyme.</a> J Biol Chem. 243, 2108-2114 (1968).</li><li>Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+immediate-early+TIS21+protein+and+the+leukemia-associated+BTG1+protein+interact+with+a+protein-arginine+N-methyltransferase.">The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase.</a> J Biol Chem. 271, 15034-15044 (1996).</li><li>Boffa, L. C., Karn, J., Vidali, G., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+NG,+NG,-dimethylarginine+in+nuclear+protein+fractions.">Distribution of NG, NG,-dimethylarginine in nuclear protein fractions.</a> Biochem Biophys Res Commun. 74, 969-976 (1977).</li><li>Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+at+a+glance.">Arginine methylation at a glance.</a> J Cell Sci. 120, 4243-4246 (2007).</li><li>Lee, Y. H., Stallcup, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+protein+arginine+methylation+of+nonhistone+proteins+in+transcriptional+regulation.">Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.</a> Mol Endocrinol. 23, 425-433 (2009).</li><li>Bedford, M. T., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Mol Cell. 33, 1-13 (2009).</li><li>Bikkavilli, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled3+is+a+novel+arginine+methyl+transferase+substrate.">Dishevelled3 is a novel arginine methyl transferase substrate.</a> Scientific reports. 2, 805 (2012).</li><li>Bikkavilli, R. K., Malbon, 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=Arginine+methylation+of+G3BP1+in+response+to+Wnt3a+regulates+{beta}-catenin+mRNA.">Arginine methylation of G3BP1 in response to Wnt3a regulates {beta}-catenin mRNA.</a> J Cell Sci. 124, 2310-2320 (2011).</li><li>Bikkavilli, R. K., Malbon, 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=Wnt3a-stimulated+LRP6+phosphorylation+is+dependent+upon+arginine+methylation+of+G3BP2.">Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.</a> J Cell Sci. , (2012).</li><li>Tini, M., Naeem, H., Torchia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+arginine+methylation+in+transcription.">Biochemical analysis of arginine methylation in transcription.</a> Methods Mol Biol. 523, 235-247 (2009).</li><li>Lee, J., Cheng, D., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+protein+methylation.">Techniques in protein methylation.</a> Methods Mol Biol. 284, 195-208 (2004).</li><li>Cheng, D., Vemulapalli, V., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+applied+to+the+study+of+protein+arginine+methylation.">Methods applied to the study of protein arginine methylation.</a> Methods Enzymol. 512, 71-92 (2012).</li><li>Zurita-Lopez, C. I., Sandberg, T., Kelly, R., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+protein+arginine+methyltransferase+7+(PRMT7)+is+a+type+III+enzyme+forming+omega-NG-monomethylated+arginine+residues.">Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues.</a> J Biol Chem. 287, 7859-7870 (2012).</li></ol>

著者らは、開示することは何もない。

本明細書に記載のプロトコルは、日常的に同定PRMT基質のメチル化状態を確立するために使用される。この強力、かつ一貫性のアッセイはまた、それらの基質のためのPRMTsの特異性に決定的な証拠を提供します。このアッセイの成功のための重要なコンポーネントは次のとおりです。哺乳動物細胞におけるPRMTsの1の発現、精製PRMTs 0002活性、基質の3発現および精製、ニトロセルロース膜へのタ?...

タンパク質のメチル化は、最初に1968年1に記載された。それは、研究者は、この翻訳後修飾2の重要性を認めるようになったことを、1996年にPRMT1の最初のクローニングまでではなかった。興味深いことに、核抽出物のタンパク質中のアルギニン残基の約​​2％は、本変形例の存在量を示す、3メチル化されている。アルギニンは、塩基性側鎖を有するアミノ酸を正に帯電している窒素/ sのアルギニンの側鎖中には、翻訳後にメチル基、アルギニンのメチル化4-6として知られているプロセスの添加によって修飾することができる。アルギニンメチル化は、酵素、すなわちクラスによって触媒される。、タンパク質アルギニンメチルトランスフェラーゼ（PRMTs）。アルギニンはモノメチル化またはジメチル化し、後者は、工程4-6を触媒PRMTsの種類に応じて、対称または非対称のいずれかであり得ることがことができます。 アルギニンを表示するタンパク質電子グリシンリッチモチーフはPRMT媒介触媒作用のための潜在的標的である。基質のPRMT媒介メチル化は、正常な細胞ホメオスタシス7-9に重要である全てが、タンパク質-タンパク質相互作用、タンパク質-核酸相互作用、タンパク質機能、遺伝子発現、および/ ​​または細胞内シグナル伝達を調節することが示されている。タンパク質アルギニンメチル化の生物学的役割を理解するために、正確で効率的かつ再現可能なアッセイを同定PRMT基質のメチル化状態を確立するために必要とされる。 それらの基質のメチル化を触媒する精製PRMTsの能力を評価するインビトロメチル化アッセイでは 、アルギニンメチル化10-12を研究するための広く受け入れられアッセイである。このアッセイの全体的な成功は、主に精製されたPRMTsの活性に依存している。 PRMTsを発現させ、細菌、または哺乳動物細胞10,11から精製することができる。 dと、このインビトロメチル化アッセイ、プロトコルセクションでetailed、もともとチニと同僚10によって記載された方法に基づいている。このプロトコルでは、詳細には哺乳動物細胞におけるPRMT1の発現および精製に関与するステップを示す。タンパク質結合タンパク質1（G3BP1）、既知のPRMT基板8を 、活性化はRas-GTPアーゼ上のメチル化を触媒する精製PRMT1の能力は、後述のようなS-アデノシル-L-[メチル- 3 H]メチオニンの存在下で評価した。メチル供与体。このアッセイを用いて、確実に、タンパク質アルギニンメチル化の研究における主要なステップであるメチ小説や既知の基質にPRMTsの能力を定義することができます。

<table border="0" cellpadding="0" cellspacing="0" width="673">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">S-adenosyl-L-[methyl-3H] methionine</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ecoscint ultra&#160;</td>
			<td>National Diagnostics</td>
			<td>LS-270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bottle top dispenser</td>
			<td>National Diagnostics</td>
			<td>LS-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoFluor</td>
			<td>National Diagnostics</td>
			<td>LS-315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 ml Scintillation Vials</td>
			<td>National Diagnostics</td>
			<td>SKU:SVC-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GST-sepharose</td>
			<td style="width:136px;">GE Life Sciences</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-Glutathione Reduced</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Lipofectamine</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:111px;"></td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Beas2B</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Optimem</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NP-40</td>
			<td style="width:136px;">US-Biologicals</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">SDS</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sodium deoxycholate</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PMSF</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">anti-HA affinity matrix</td>
			<td style="width:136px;">Roche</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Tris</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nacl</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bio-rad gel doc</td>
			<td style="width:136px;">Bio Rad</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">anti-HA antibody</td>
			<td style="width:136px;">roche</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Ponseau S</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro methylation assay to study protein arginine methylation

タンパク質アルギニンメチル化は、核内で最も豊富な翻訳後修飾の一つである。タンパク質アルギニンメチル化は、識別および/または決定プロテオミクスのアプローチを介して、および/またはメチル-アルギニン特異的抗体を用いたイムノブロッティングすることができる。しかしながら、これらの技術は、時には誤解を招くことがあり、しばしば、偽陽性の結果を与えることができる。最も重要なことは、これらの技術は、PRMTの基質特異性を支持する直接的な証拠を提供することができない。 インビトロメチル化アッセイ、一方、敏感である有用な生化学的ア ​​ッセイであり、同定されたタンパク質が実際にPRMT基質である場合には一貫して明らかにした。典型的なインビトロメチル化アッセイは、精製され、アクティブPRMTs、精製された基板と、放射性同位体標識されたメチル供与体（S-アデノシル-L-[メチル- 3 H]メチオニン）が含まれています。ここでは、触媒的に活性なPRMT1、普遍的に発現PRMTファミリーメンバーを単離するためにステップバイステップのプロトコルを記述します。私に精製PRMT1のメチルトランスフェラーゼ活性は、後でS-アデノシル-L-メチル供与体としての[メチル- 3 H]メチオニンの存在下で、タンパク質1（G3BP1）に結合Rasは、GTPアーゼ活性化タンパク質、既知のPRMT基板上に試験した。このプロトコルは、新たな生理PRMT1基質のメチル化状態を確立するためだけでなく、タンパク質アルギニンメチル化の基本的なメカニズムを理解するためだけでなく、使用することができる。

<html> GST-G3BP1をメチル化するためにHA-PRMT1の能力は、インビトロメチル化アッセイによって決定した。 Beas2B細胞溶解物から精製されたHAタグ付きPRMT1をメチル供与体として、GST-G3BP1、および1 S-アデノシル-L-のμCiの[メチル- 3 H]メチオニンを含むメチル化反応で使用した。 PRMT1を効率的に、GST-G3BP1（ 図1、上のパネル）のメチル化を触媒することができます。空のベク?...

Watch this Scientific Journal Video about In vitro Methylation Assay to Study Protein Arginine Methylation at JoVE.com

In vitro Methylation Assay to Study Protein Arginine Methylation

タンパク質アルギニンメチル化酵素、すなわちクラスによって触媒。、タンパク質アルギニンメチルトランスフェラーゼ（PRMTs）は、タンパク質内のアルギニンのメチル基の酵素添加の過程である。 インビトロメチル化アッセイは、既知または新規のPRMT基質のメチル化状態を評価するための最も信頼できるツールである。

タンパク質アルギニンメチル化を研究するためのin vitroでメチル化アッセイで

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

in-vitro-methylation-assay-to-study-protein-arginine-methylation

Research

JoVE Journal

Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.1K ビュー.  University of Illinois at Chicago. タンパク質アルギニンメチル化酵素、すなわちクラスによって触媒。、タンパク質アルギニンメチルトランスフェラーゼ（PRMTs）は、タンパク質内のアルギニンのメチル基の酵素添加の過程である。 インビトロメチル化アッセイは、既知または新規のPRMT基質のメチル化状態を評価するための最も信頼できるツールである。 

ビデオ: In vitro Methylation Assay to Study Protein Arginine Methylation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

のインプリント遺伝子のDNAメチル化の定量シロイヌナズナ胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

タンパク質のメンブレンオーバーレイアッセイ：可溶性および不溶性タンパク質間の相互作用をテストするためのプロトコル in vitroで</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNAメチル化：重亜硫酸修飾と分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

の適用<em&gt;インビトロ</emDPSタンパク質のストレス調停プロパティを可視化するために&gt; DNA保護アッセイ

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

のための内皮細胞チューブ形成アッセイ<em&gt;体外</em&gt;血管新生に関する研究

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

塩基対解像度でDNAメチル化の評価のための強化されたの減少表現重亜硫酸塩シークエンシング

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.