This work was supported by NIH grants (NS070577 and NS084869).

注：以下の引用された全ての溶液の組成は、材料/機器の表に記載されています。 1.プロテインAカラム抗体精製<ol><li>手順を開始する前に15分間室温にプロテインAスピンカラムと、必要なすべてのバッファーを平衡化します。 </li><li>結合緩衝液プロテインAのIgGと1：免疫血清1を希釈します。 （血清5mlをお勧めします）。 </li><li>静かにキャップ内に提出することができる任意の樹脂を除去するために、ベンチトップ上のタンパク質スピンカラムをタップします。トップキャップを外し、ゆっくりと底部の閉鎖をスナップオフ。 15ミリリットルのコレクションチューブにカラムを置き、ストレージソリューションを排出することができます。 </li><li>結合緩衝液プロテインAのIgGの5ミリリットルを加え、溶液を排出させます。 </li><li>カラムに希釈した免疫血清を適用し、フロースルーを収集します。最良の結果を得るために、カラムの抗体結合capacitの80％未満の抗体の濃度が含まれているサンプル容量を追加Y。 </li><li> 280 nmでの吸光度が0.1以下になるまで、プロテインAのIgG 15mlの結合緩衝液またはでカラムを洗浄します。 </li><li> 5標識されたコレクションチューブに中和緩衝液100μlを追加します。 </li><li>プロテインAカラムへのIgG溶出緩衝液5mlを加え、中和緩衝液100μlを含むチューブのそれぞれに1mlの画分を収集します。 </li><li> 0.5より大きい吸光度280 nmおよびプールの画分で各画分の吸光度を測定します。透析の準備ができるまで氷上でプールした画分を保管してください。 </li><li> IgGを溶出緩衝液8mlのを追加することで、列を再生成し、溶液がカラムを通って流れることを可能にします。 </li><li>溶離液のpHは7.4に戻るまで、プロテインAのIgG結合液でカラムを洗浄します。 </li><li>貯蔵溶液5ml（PBS中0.02％アジ化ナトリウム）を添加することにより、カラムを保存。約3mlの列、キャップ底部に残り、トップキャップを固定した場合。 4℃で列を保管してください。 </li><li>長さOを決定製造業者の指示に従って必要なF透析チューブ。 16ミリメートルに平らな直径のチューブを使用すると、透析される溶液1ml当たり、チューブの長さの5.47センチメートルを使用しています。チューブをカットし、15〜30分間のPBS（pH7.4）ですすいでください。 </li><li>倍と透析チューブの一端をクリップ、PBSのpHは7.4で平衡化した透析チューブに含有画分をプールしたIgGを転送し、もう一方の端を閉じます。 </li><li> 2時間pH7.4のPBSの200ボリュームに対して室温で透析します。 </li><li>透析緩衝液（新鮮なPBS pH7.4）に変更し、室温でさらに2時間透析。 </li><li>透析緩衝液（新鮮なPBS pH7.4）に変更し、4℃で一晩透析します。 </li><li>さらなる精製工程を続行する準備ができるまで4℃で透析した抗体溶液を保管してください。 </li><li> 280 nmでの吸光度を用いて抗体濃度と21万M -1 cm -1で12の280nmにおける抗体のモル吸光係数を評価します。 </li></ol>アミノリンクカップリング樹脂にLBDタンパク質の2カップリング<ol><li> 2列、Nur77ためNor1のための1つおよび他を準備します。両方のタンパク質のための同じプロトコルに従ってください。 </li><li>氷上で樹脂に結合するタンパク質を解凍します。 280 nmの12で、そのモル吸光係数とその吸光度を用いてタンパク質濃度を決定します。 <ol><li>あるいは、そのようなビシンコニン酸アッセイ13のようなタンパク質の決意アッセイを使用しています。タンパク質は、アミン含有緩衝透析またはカップリングバッファーに対してバッファー交換中に溶解されている場合。タンパク質は、適当な緩衝液（無遊離アミン）である場合のカップリング緩衝液で4倍に希釈します。 </li></ol></li><li>タンパク質2mgのための樹脂の2ミリリットルを使用してください。以降のすべての手順は、10ミリリットルの列に2ミリリットルの樹脂のためのものです。タンパク質の最大10 mgの（1：1スラリー1を2ml）樹脂を1mlあたり連結することができます。 </li><li>底部にフリットを押し、それを介してPBS pHが7.4を実行して、10ミリリットルのカラムを準備します。キャップT彼は塔の底部とのカップリング緩衝液2ミリリットルを追加します。 </li><li> 、カラムへのスラリーの所望の体積（樹脂の2ミリリットルを得るために4ミリリットル）を追加底部キャップを除去し、カラムの流出をしてみましょう。カップリング緩衝液の6ミリリットル（3樹脂床容積）の合計でカラムを洗浄し、カラムを排水することができます。カップリング緩衝液を排出した後、ボトムキャップを交換してください。 </li><li> （280nmでの吸光度またはビシンコニンアッセイのいずれかを使用してステップ2.11で溶出液のタンパク質濃度を比較することにより、結合効率を評価するために、タンパク質溶液のアリコートを保持）カラムへの結合緩衝液に懸濁し、タンパク質2-4 mlを加え。キャップと均一な溶液を持つように転倒混和します。 </li><li>空の1.8ミリリットルマイクロチューブを秤量し、ドラフトに移動します。慎重に予め秤量したチューブ中のNaCNBH 3（約0.05g）を転送します。チューブを閉じ、のNaCNBH 3を含むチューブの重量を量ります。フードの外管を開けないでください。 </li><li>の重量を基準にしてチューブに移しのNaCNBH 3は 、1 M NaOHを添加することによって5 M溶液を作成します。のNaCNBH 3の分子量は、62.84グラム、従って0.05グラムは、（0.05 / 62.84 = 7.96×10 -4モル）を7.96×10 -4モルに等しいです。 5 M溶液を作製するには、（7.96×10 -4 / 5）1 M NaOHを159μlを添加します。 </li><li>考慮に樹脂容量を取る反応の総容量を測定します。反応の1ml当たり5 MのNaCNBH 3の10μLを加えます。 <ol><li>たとえば、次の4ミリリットルの反応ボリュームの5 MのNaCNBH 3の40μlを添加します。樹脂1mlのコメントを追加しましたタンパク質の3ミリリットルのために、総容量を4 mlです。 </li></ol></li><li>カラムに蓋をし、転倒混和します。チューブ回転で列を固定し、反応物を4℃で一晩続けましょう。 </li><li>午前中は、化学フードに列を持参し、いくつかのガスが形成されている可能性がありますように慎重にキャップを取り外します。きれいなチューブにカラムを排水し、タンパク質コンを測定するために、溶出液を保存セント280nmの方法12またはビシンコニン酸アッセイでの吸光度を用いて、ステップ2.6で出発タンパク質濃度と比較します。 </li><li>カップリング緩衝液4mlで樹脂を洗浄し、カラムの流出をしてみましょう。 </li></ol> 3.残りのサイトをブロック<ol><li>クエンチング緩衝液4mlで樹脂を洗浄。ドレインと底部キャップを交換してください。急冷2mlの緩衝液を添加し、樹脂を中断。 </li><li>樹脂容量と消光バッファ量を測定することにより、総反応容量は、その後、反応容量1mlあたり5 MのNaCNBH 3の10μlを添加する評価します。 </li><li> 30分転倒、室温で穏やかに混合します。 30分後、ドラフト内で列をもたらします。慎重にトップを削除してから、底部キャップを除去し、廃棄物の管に、カラムの流出をしてみましょう。 </li><li>洗浄溶液の5樹脂ボリュームでカラムを洗浄します。溶出液のabsoを測定することにより、残留タンパク質の存在のための洗浄を監視280nmでrbance。非結合タンパク質を、高塩濃度によって洗い流されます。 </li><li> 0.02％アジ化ナトリウムを含有する脱気したPBSのpHが7.4の6ミリリットルで樹脂を洗浄。必要になるまで4℃で列を保管してください。 </li></ol> Nurr1を特異的抗体の4精製<ol><li>すすぎNur77 LBDは、pH7.4のPBS 10mlでカラムに結合し、カラムの流出をしてみましょう。カラムの底部に蓋をして、新しい15ミリリットルのコレクションチューブに排出さNur77 LBD結合列を配置します。 </li><li> 、タンパク質5mlの精製抗のNurr1抗体を追加し、1時間4℃で回転体の列と場所をキャップ。上部と下部のキャップを外し、フロースルーを収集します。 Nor1のLBD結合列に追加する直前まで、氷上に置いておきます。 </li><li>すすぎNor1のLBDは、pH7.4のPBS 10mlでカラムに結合し、カラムの流出をしてみましょう。カラムの底部に蓋をし、15 mlコレクションチューブNor1のLBD結合列を配置します。 </li><li> Nur77 LBDのクーデターからの流れを介して追加4℃で1時間回転器でのカラムと場所をキャップ、列を主導しました。上部と下部のキャップを外し、フロースルーを収集します。 </li><li> 280nmでの吸光度を測定することにより抗体濃度を測定します。 </li></ol>精製抗のNurr1抗体の5 ELISAに基づく分析<ol><li> 96ウェルプレートのウェルにタンパク質を100μl（2.5μgの/ ml）を追加します。 3種類のタンパク質をテストする場合は、H4のウェルA1、H8にウェルA5へのタンパク質2を100μl、およびH12のウェルA9へのタンパク質3の100μlにタンパク質1の100μlを添加します。プレートをカバーし、4℃で一晩インキュベートします。 </li><li> pH7.4のPBSのウェル当たり300μlの二回コーティングしたウェルを洗浄し、抗原全体コーティングされたウェルにELISAブロッキング緩衝液の300μlを添加し、室温で2時間インキュベートします。 </li><li>プレートは、精製された抗のNurr1抗体の所望の出発希釈を準備ブロックしているときにELISAブロッキング緩衝液中（10〜100μg/ mlの範囲）。 </li><li>ブロッキングの2時間後、全てのウェルに新鮮なELISAブロック緩衝液100μlを用いたELISAブロックバッファを交換します。 </li><li>行Aの全てのウェルに希釈した精製された抗のNurr1抗体の100μlを添加する逐次Cを行ごとにBから溶液100μlを転送することにより、続いてB列にA列からウェルに100μlの溶液を転送することにより、抗体を希釈余分な100μLを捨てる行Gのウェル中の溶液を混合した後G.行ダウン続けます。行Hのウェルは、緩衝液のみをブロックの最初の100μLを受けます。 1時間室温でインキュベートします。 </li><li> pH7.4のPBSでプレートを0.05％のTween 20のウェル当たり300μlので3回洗浄し、過剰な洗浄緩衝液を除去するためにプレートをブロット。 </li><li>ホースラディッシュペルオキシダーゼ（HRP）ELISAブロック緩衝液で希釈したコンジュゲートヤギ抗ウサギIgGの100μlを加える（1：8,000希釈し、典型的な範囲は、1：25,000：5,000 1）。 1時間室温でインキュベートします。 </li><li>プレート番目を洗いますREE（3）5.6倍に記載の方法。脱イオン水300μlのとそれらを充填することにより、全てのウェルを洗浄します。紙を吸収する上でプレートを反転させて余分な水分を吸い取ります。 </li><li> &#39;、5,5&#39; 3,3の100μlを添加しテトラメチルベンジジン（室温に温め）を全てのウェルに、室温で暗所で15〜30分間インキュベートします。 </li><li> SO 4を 2 NH 2の50μLを加えて反応を停止します。 </li><li> 650 nmでのバックグラウンドを差し引い450 nmの吸光度を読みます。 </li></ol>

<ol>
	<li>Hawk, J. D., Abel, T. <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+NR4A+transcription+factors+in+memory+formation.">The role of NR4A transcription factors in memory formation.</a> Brain Res. Bull. 85 (1-2), 21-29 (2011).</li><li>Law, S. W., Conneely, O. M., DeMayo, F. J., O'Malley, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+brain-specific+transcription+factor,+NURR1.">Identification of a new brain-specific transcription factor, NURR1.</a> Mol. Endocrinol. 6 (12), 2129-2135 (1992).</li><li>Zetterstr&#246;m, R. H., 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=Dopamine+neuron+agenesis+in+Nurr1-deficient+mice.">Dopamine neuron agenesis in Nurr1-deficient mice.</a> Science. 276 (5310), 248-250 (1997).</li><li>Kadkhodaei, B., 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=Nurr1+is+required+for+maintenance+of+maturing+and+adult+midbrain+dopamine+neurons.">Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons.</a> J. Neurosci. 29 (50), 15923-15932 (2009).</li><li>Decressac, M., Volakakis, N., Bj&#246;rklund, A., Perlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NURR1+in+Parkinson+disease--from+pathogenesis+to+therapeutic+potential.">NURR1 in Parkinson disease--from pathogenesis to therapeutic potential.</a> Nat Rev Neurol. 9 (11), 629-636 (2013).</li><li>Pe&#241;a de Ortiz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+Expression+of+the+Orphan+Nuclear+Receptor+Gene+hzf-3/nurr1+during+Spatial+Discrimination+Learning.">Hippocampal Expression of the Orphan Nuclear Receptor Gene hzf-3/nurr1 during Spatial Discrimination Learning.</a> Neurobiol Learn Mem. 74 (2), 161-175 (2000).</li><li>Vecsey, C. G., 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=Histone+deacetylase+inhibitors+enhance+memory+and+synaptic+plasticity+via+CREB:CBP-dependent+transcriptional+activation.">Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation.</a> J. Neurosci. 27 (23), 6128-6140 (2007).</li><li>Col&#243;n-Cesario, W. I., 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=Knockdown+of+Nurr1+in+the+rat+hippocampus:+implications+to+spatial+discrimination+learning+and+memory.">Knockdown of Nurr1 in the rat hippocampus: implications to spatial discrimination learning and memory.</a> Learn Mem. 13 (6), 734-744 (2006).</li><li>McQuown, S. C., 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=HDAC3+is+a+critical+negative+regulator+of+long-term+memory+formation.">HDAC3 is a critical negative regulator of long-term memory formation.</a> J. Neurosci. 31 (2), 764-774 (2011).</li><li>Hawk, J. D., 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=NR4A+nuclear+receptors+support+memory+enhancement+by+histone+deacetylase+inhibitors.">NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors.</a> J. Clin. Invest. 122 (10), 3593-3602 (2012).</li><li>Bridi, M. S., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NR4A+orphan+nuclear+receptors+mediate+transcription-dependent+hippocampal+synaptic+plasticity.">The NR4A orphan nuclear receptors mediate transcription-dependent hippocampal synaptic plasticity.</a> Neurobiol Learn Mem. 105, 151-158 (2013).</li><li>Gill, S. C., von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+protein+extinction+coefficients+from+amino+acid+sequence+data.">Calculation of protein extinction coefficients from amino acid sequence data.</a> Anal. Biochem. 182 (2), 319-326 (1989).</li><li>Smith, P. 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Zetterstr&#246;m, R. H., Williams, R., Perlmann, T., Olson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+the+immediate+early+transcription+factors+Nurr1+and+NGFI-B+suggests+a+gene+regulatory+role+in+several+brain+regions+including+the+nigrostriatal+dopamine+system.">Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system.</a> Brain Res. Mol. Brain Res. 41 (1-2), 111-120 (1996).</li><li>Xiao, Q., Castillo, S. O., Nikodem, V. M. <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+messenger+RNAs+for+the+orphan+nuclear+receptors+Nurr1+and+Nur77+(NGFI-B)+in+adult+rat+brain+using+in+situ+hybridization.">Distribution of messenger RNAs for the orphan nuclear receptors Nurr1 and Nur77 (NGFI-B) in adult rat brain using in situ hybridization.</a> Neuroscience. 75 (1), 221-230 (1996).</li></ol>

著者らは、開示することは何もありません。

このプロトコルの成功は、目的のタンパク質ファミリーの特定のメンバーに対して産生された抗体の特徴付けのための純粋なタンパク質の利用可能性に依存しています。それは明らかにELISAおよびウェスタンブロッティングによる有意な交差反応性があることが確立されるまで、プロテインA / Gを使用して抗体を精製する必要はありません。 本文で述べたように、このよ?...

転写因子のNurr1およびその同族体（Nur77およびNor1のは）核内受容体サブファミリー4A（NR4A）1に属します。それらの内因性リガンドはまだ同定されていないので、彼らはまた、オーファン受容体です。 Nurr1を最初は1992年にクローン化され、2脳内で発現することが知られているが、中脳ドーパミンニューロンの発達および維持のための重要な役割は、ノックアウトマウスの研究により明らかになった3。また、中脳ドーパミンニューロンの維持のための重要な役割は、最近、条件付きノックアウト研究4によって実証されました。による中脳ドーパミンニューロンのためのNurr1の重要な役割を示すこれらのエレガントな研究に、多くのその後の研究は、主に中脳ドーパミンニューロンおよびドーパミン関連の神経変性疾患、パーキンソン病（PD）に焦点を当ててきた5。 注目すべきは、Nurr1をは中脳ドーパミン（MDA）ニューロンにおいてだけでなく、ディだけでなく表現されていますそれは強くNurr1を、様々な脳機能に重要な役割を果たしていることを示す最近の研究によって支持されている多くの非DAの分野で機能的な役割を有し得ることを示唆している詩の脳領域2、。例については、このような学習として、メモリ誘導活性を示された、またはその他の海馬依存タスクがアップレギュレーション海馬6,7中のNurr1の発現をもたらします。また、海馬でのNurr1発現のノックダウンが強くのNurr1は、多くの脳領域で多様な役割を果たしていることを示唆し、長期記憶および/ ​​またはシナプス可塑性8-11を損なうのに十分でした。したがって、さらにNurr1をの細胞型特異的および細胞内発現を理解するためには、そのホモログNur77またはNor1のに任意の交差反応性を示さないのNurr1特異的抗体を使用することが望ましいです。本稿では、Nurr1を特異的抗体を生成し、その特異性を示す追加データを提示するために事前に吸着プロトコルについて説明します。

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	<tbody>
		<tr>
			<td>Purified Ligand Binding Domain from Nurr1, Nor 1 and Nur77</td>
			<td></td>
			<td></td>
			<td>Column Storage solution</td>
		</tr>
		<tr>
			<td>AminoLink Coupling Resin and Kit</td>
			<td>Thermo Scientific</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A spin column</td>
			<td>Thermo Scientific</td>
			<td>89978</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Flate-bottom plates, High Flange, 330&#956;L</td>
			<td>Thermo Scientific</td>
			<td>3455</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>KPL</td>
			<td>50-675-69</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk Diluent/ Blocking Concentrate&#160;</td>
			<td>KPL</td>
			<td>50-82-01</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Elution Buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1 M Tris-HCI Buffer, pH 7.5</td>
			<td>Life Technologies</td>
			<td>15567-&#173;&#8208;027</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Radish Peroxidase conjugated Goat anti-Rabbit IgG (H+L)</td>
			<td>Thermo Scientific</td>
			<td>31460</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Resin</td>
			<td>Thermo Scientific</td>
			<td>88221</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;, 5, 5&#39; Tetramethylbenzydine</td>
			<td>Thermo Scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>2N H2SO4</td>
			<td>Macron Fine Chemicals</td>
			<td>H381 05</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A IgG Binding Buffer</td>
			<td>Thermo Scientific</td>
			<td>21001</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Elution Buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutralization Buffer</td>
			<td></td>
			<td></td>
			<td>1M Tris-&#173;&#8208;HCl pH 8.5</td>
		</tr>
		<tr>
			<td>Column Storage solution</td>
			<td></td>
			<td></td>
			<td>Phosphate Buffered Saline containing 0.02% sodium azide</td>
		</tr>
		<tr>
			<td>Spectra/Por 7 pre-treated dialysis tubing</td>
			<td>Spectrum Labs</td>
			<td>132128</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Disposable columns</td>
			<td>Thermo Scientific</td>
			<td>29924</td>
			<td></td>
		</tr>
		<tr>
			<td>AminoLink Coupling Buffer</td>
			<td></td>
			<td></td>
			<td>0.1M sodium phosphate, 0.05% NaN3, pH 7.0</td>
		</tr>
		<tr>
			<td>Quenching buffer</td>
			<td></td>
			<td></td>
			<td>1M Tris. HCI, 0.05% NaN3, pH 
			7.4</td>
		</tr>
		<tr>
			<td>Wash Solution</td>
			<td></td>
			<td></td>
			<td>1M NaCl, 0.05% NaN3</td>
		</tr>
		<tr>
			<td>ELISA Blocking buffer</td>
			<td></td>
			<td></td>
			<td>1% Normal Horse serum (NHS), 1% Normal Goat Serum (NGS) in 1X KPL milk diluent</td>
		</tr>
		<tr>
			<td>Storage Solution</td>
			<td></td>
			<td></td>
			<td>PBS pH 7.4 containing 0.02% sodium azide</td>
		</tr>
		<tr>
			<td>Micropipettes: 10 &#956;l; 20 &#956;l; 200 &#956;l; 1000 &#956;l</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson&#8217;s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

Nur77 LBDとNor1のLBDのカラムに通すことによって、その後のタンパク質の比較タンパク質とカラム精製の​​Nurr1に特異的な抗体精製抗Nurr1を抗体は、図1に示されている。図から分かるように、プロテインA精製の​​Nurr1抗体は強い結合を示しましたNurr1をLBDに。しかし、それはまた、Nur77 LBDへとNor1のLBDへの有意な結合を示しました。プロテインA精製のNurr1抗体はさらにNur77 LBDおよ?...

Watch this Scientific Journal Video about Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77 at JoVE.com

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

NURR-1特異的ポリクローナル抗体の密接な相同体に対する交差反応性の自由、Nor1のとNur77の生産

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The ...

production-nurr-1-specific-polyclonal-antibodies-free-cross

Research

JoVE Journal

Biology

7.4K ビュー.  McLean Hospital. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

ビデオ: Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

部位特異的リコンビナーゼ（SSR）技術の原理

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

生物学

Operant Learning of Drosophila at the Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

トルクメーターでのショウジョウバエのオペラント学習

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

ハニービーの嗅覚学習中のキノコ体の神経細胞のイメージング- 生体内のCa 2 + の

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

細胞毒性の継続とリアルタイム監視のための自律的に生物発光哺乳類細胞

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

ショウジョウバエにおける温度嗜好挙動とその概日リズムの設計と解析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

使用モデルタンパク質への非標準アミノ酸の残基特異的取り込み<em&gt;エシェリヒア・コリ</em&gt;無細胞転写翻訳システム

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 &#176;C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding &#39;tuft.&#39;&#160;Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Kuga et al. discovered that phosphorylation-status specific antibodies against the actin binding protein girdin phosphorylated at tyrosine 1798 (pY1798) can be used to label tuft cells (TCs). This protocol allows robust visualization of TCs using immunofluorescent staining of free-floating jejunum cryosections with pY1798 antibodies.

フローティング マウス腸凍結切片で腸内房細胞を可視化する抗リン girdin 抗体の使用

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

アグロバクテリウム-媒介遺伝子変換、トランスジェニック産生、および米における男性生殖発達の研究への応用

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

全種サイズ樹脂断面の簡易ドライ断面化方法とその応用

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

細菌無細胞リザートの迅速、手頃な価格、および単純な生産

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, ...