私たちは、財政支援のために、次の感謝したいと思います：BBSRC研究所戦略計画の資金調達 - CF＆TMW。 BBSRC東バイオDTP資金 - LG。エジンバラのダーウィン·トラスト - MLH。我々はまた、この原稿にTREM2最適化を含めるための許可を博士バリーMcCollに感謝したいと思います。

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Biotechnology. 1992 (24), 145-149 (1979).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Protocols on CD-ROM. , (1998).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Curr Protoc Mol Biol. 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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=Comparison+of+Stain-Free+gels+with+traditional+immunoblot+loading+control+methodology.">Comparison of Stain-Free gels with traditional immunoblot loading control methodology.</a> Anal Biochem. 430 (2), 108-110 (2012).</li><li>Zellner, M., 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=Fluorescence-based+Western+blotting+for+quantitation+of+protein+biomarkers+in+clinical+samples.">Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.</a> Electrophoresis. 29, 3621-3627 (2008).</li><li>Kielar, 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=Molecular+correlates+of+axonal+and+synaptic+pathology+in+mouse+models+of+Batten+disease.">Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.</a> Hum Mol Genet. 18, 4066-4080 (2009).</li><li>Mutsaers, C. A., Lamont, D. J., Hunter, G., Wishart, T. M., Gillingwater, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+proteomics+identifies+Calreticulin+and+GRP75/Mortalin+as+peripherally+accessible+protein+biomarkers+for+spinal+muscular+atrophy.">Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.</a> Genome Med. 5 (10), 95 (2013).</li><li>Wishart, T. M., 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=Dysregulation+of+ubiquitin+homeostasis+and+&#946;-catenin+signaling+promote+spinal+muscular+atrophy.">Dysregulation of ubiquitin homeostasis and &#946;-catenin signaling promote spinal muscular atrophy.</a> J Clin Invest. 124 (4), 1821-1834 .</li><li>Mathews, S. T., Plaisance, E. P., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+systems+for+westerns:+chemiluminescence+vs.+infrared+detection.">Imaging systems for westerns: chemiluminescence vs. infrared detection.</a> Methods Mol Biol. 536, 499-513 (2009).</li><li>Hawes, J. J., Brunzell, D. H., Wynick, D., Zachariou, V., Picciotto, 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=GalR1,+but+not+GalR2+or+GalR3,+levels+are+regulated+by+galanin+signalling+in+the+locus+coeruleus+through+a+cyclic+AMP-dependent+mechanism.">GalR1, but not GalR2 or GalR3, levels are regulated by galanin signalling in the locus coeruleus through a cyclic AMP-dependent mechanism.</a> J Neurochem. 93 (5), 1168-1176 (2006).</li><li>Bond, D., Primrose, D. A., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+signaling+events+in+Drosophila+S2+cells.">Quantitative evaluation of signaling events in Drosophila S2 cells.</a> Biol Proceed Online. 10 (1), 20-28 (2008).</li><li>Wishart, T. M., 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=Differential+proteomics+analysis+of+synaptic+proteins+identifies+potential+cellular+targets+and+protein+mediators+of+synaptic+neuroprotection+conferred+by+the+slow+Wallerian+degeneration+(Wlds)+gene.">Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.</a> Mol Cell Proteomics. 6, 1318-1330 (2007).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+western+in+town:+a+contemporary+twist+on+the+classic+western+blot+analysis.">The fastest western in town: a contemporary twist on the classic western blot analysis.</a> J. Vis. Exp. (84), 51149 (2014).</li></ol>

The authors have nothing to disclose.

配慮と計画は、あらゆる実験に先立って不可欠であり、最終的に使用されるテクニックの成功を決定することができます。使用するために適切な抗体、転送および可視化方法を選択しようとすると、WBを使用したタンパク質検出の進歩は、潜在的な障害ブロックの過多を提示することができます。幸いなことに、タンパク質の存在とサンプル間ますます微妙な発現差を決定するために日常的に使用することができるQFWB注意深くチェックし、適切な対策を使用する。このプロトコルは、回避し、および/またはそれに関連する多くの一般的な落とし穴のいくつかを克服するために、蛍光定量的ウェスタンブロッティングと同様に、いくつかのトラブルシューティングの戦略の包括的なガイドを提供します。 感度を維持し、真の定量化と比較可能な測定値を得るために用いられる重要なステップは次のとおり組織サンプルから1）堅牢なタンパク質抽出する。 2）試料調製。 3）正確なタンパク質Loadinのgの全タンパク質分析によって決定。 4）I-ブロットを使用して、タンパク質の最適な転送を、赤外線イメージャと関連ソフトウェアを使用して、0.1％のTween20を含むブロッキング緩衝液中で一次および二次抗体の5）準備、および6）が正しいの可視化と分析。 赤外蛍光検出本当に定量的であり、より伝統的なECL検出技術3,11に比べてより高い感度を提供する。この検出システムは、多面的であり、そのようなものとしてQFWBに限定されるものではない。このシステムは、全体の組織切片12の可視化および定量化を可能にする、低電力での免疫組織学的標識イメージングが可能である。これは定量的評価の点で従来の顕微鏡では、従来の免疫蛍光の取り込みに匹敵する遠赤色イメージングを見ることができ、解像度の点で潜在的な将来の発展の一つの領域である。 しかし、Pの微妙な変化に対してより大きな感度を有するそれは可変性を確保するために重要であるrotein式が最小に維持され、制御措置が強固なプロトコルに厳しい。検出は実数および転送する点で、製造業者のガイドラインをテストする場合、これは決定するために、サンプル負荷が均一であることの保証、一次および二次抗体の最適化を提供するために全タンパク質染色ゲルの生成に続いて、組織サンプルからの厳密なタンパク質の抽出から始まる時間効率的なタンパク質伝達を得る。 それにもかかわらず、WBのための条件が最適化されている場合でも、まだ問題は完全にここで検討されていない可能性が西部劇を実行しているとそこに関連することがあります。これらには、タンパク質の可溶化および抽出緩衝液の選択などの要因に限定されるものではない。いくつかのバッファは、タンパク質濃度アッセイに干渉する可能性があり、いくつかの組織は、このような自動化されたmaceraの使用などのより堅牢な技術を必要とする、可溶化することが特に困難であるティンは、マックの解離とともに、Mチューブなどの容器を密封した。加えて、-80℃で抽出し、抽出されない材料の保存のための簡単​​な対策は直ちに抽出した後、最適な標識を取得し、悪い結果を週後の差を有することができる。 現代のQFWBの方法は、タンパク質発現の微妙な違いをキャプチャするためのより高感度であることが証明されており、そのようなECLなどの従来の技術と比較すると、より汎用性の同時二重標識3を許可している。これは、ウェスタンブロッティングプロトコルは、堅牢かつ正確な定量、統計分析のために容易に再現可能であることが不可欠です。このプロトコルは、高感度で、異なる組織サンプルおよび種3、様々横切っタンパク質の検出のために日常的に使用されるのに十分に堅牢であり、同じQFWB内の低および高存在量タンパク質の定量化を可能にする、従って消耗使用量を削減するだけでなく、実験の1時間あたりの1また、この技術の感度の増大は、ますます人気の検証ができます- 。OMICの研究9,14をただし精度が重要であり、適切な管理措置を含めることは、それによって誤ったデータ収集を回避するために付着されなければならない。

ウェスタンブロット（WB）は、本来、組織ホモジネート1として、複雑な生物学的サンプル中の目的のタンパク質の存在または非存在を決定するために、1970年代後半に開発された分析技術である。一般的に起因するキーの抗体 - 抗原相互作用のために、タンパク質イムノブロットと呼ばれる、方法論は、5つの別個のステップで構成されます。それらの等電点によるタンパク質の1）電気泳動分離。 2）ニトロセルロースまたはポリビニリデンジフルオリド（PVDF）膜への転移; 3）目的のタンパク質に特異的な一次抗体を用いて標識する。 4）一次抗体に対する二次抗体とのインキュベーション。 5）可視化。 可視化手法は、安全性および感度を改善するための時間と進化してきた。第WBsをのいくつかは、より多くの広く使用されている化学発光（ECL）法、次に比色とに進行放射標識タグを用いて行った。 Radioactivi比色およびECL方法論は、例えばアルカリホスファターゼまたはストレプトアビジン西洋ワサビペルオキシダーゼ（HRP）2のような酵素レポーターとの間接標識技術を使用し、一方、tyが直接特定の抗原のためのプローブに標識した。色素原または発光生成物の標識化の強度は、強いまたは弱い信号強度は、サンプル中の目的のタンパク質の多かれ少なかれ存在を示すことにより、デンシトメトリーを用いて測定される。 ECLは、最初に、続いて3を設置し、より洗練されたデジタル画像化技術を用いてX線フィルム上で開発された、より敏感なので好ま方法2が、すべての3つの方法である。 WBのデジタル画像の進歩のみならず、興味のあるタンパク質の存在または非存在を決定するために研究を可能にしたが、他のサンプルと比較したとき、選択されたタンパク質の発現レベルの推定を可能にし、したがって、と呼ぶことができる「半定量的＆＃8221 ;.測定された蛍​​光のレベルを直接サンプル内の数量と単一のタンパク質の発現に関連させる最近、真に定量的かつより高感度のウェスタンブロッティング技術が開発された：定量（QFWB）ウェスタンブロッティング蛍光。 ECL標識とQFWBを比較した場合、蛍光二次抗体の使用は、線形検出プロファイル4を生成する。これは、信号の直線性は、一般に、遍在的に発現されるハウスキーピング遺伝子5,6と、すなわち 、直接タンパク質の発現に関連した5μgの信号飽和以下の低いタンパク質負荷で発生するECL技術とは対照的である。この格差は、最も可能性の高い潜在的な信号飽和の可能性が高く、その結果、ビオチン化二次的に結合するアビジンECL基質についての利用可能な結合部位より多くによって引き起こされる。これは、ONLと呼ばれているのECLベースイムノブロット法の主な理由の一つですyは「半定量的」7。信号の飽和点は、発現レベルの微妙な差を測定する際に非常に重要であると不正確な測定をもたらすことができる。近年、増え続ける感度と微妙な表情差の識別を詳述広範なプロテオミクス技術の出現は、検証実験8,9のために真に定量的なウェスタンブロッティング上で増え続けるの依存度をもたらした。 、敏感な堅牢かつ真に定量的な方法論の適用が故に重要である。 多くの &quot;最適化&quot;ウェスタンブロッティングの方法論は、頻繁に設定したり、原因の正式な文書化されたプロトコルでは明らかではないかもしれません微妙な方法論的な調整に複製することは困難証明する独立した研究所によって利用されてきた。これはQFWBための確立された堅牢なプロトコルであり、さらに一般的な問題のthaのトラブルシューティングに貴重な戦略を提供していますtは実装時に発生する可能性がある。 このプロトコルは、もともとマウス脳ホモジネートの使用に最適化されたが、その後、組織サンプルおよび種4,9,10の広い範囲にわたって効果的に使用されてきた。特定の問題のトラブルシューティングに必要な潜在的なプロトコルのバリエーションが含まれています。

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:427px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:224px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">RIPA Buffer</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10230544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">M Tubes</td>
			<td style="width:255px;">Miltenyi Biotec Inc.</td>
			<td>130-093-236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:427px;">iBlot Transfer Stack, PVDF Regular</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>IB401001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MagicMark XP Western Protein Standard (20-220 kDa)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5602</td>
			<td>Use in gel 1 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">SeeBlue Pre-stained protein standard</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5625&#160;</td>
			<td>Use in gel 2 Total protein stained gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAge LDS Sample buffer 4X</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20X)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">PHOSPHATE BUFFERED SALINE TABLET,*TRU-ME, PHOSPHATE BUFFERED SALINE TABLET</td>
			<td style="width:255px;">Sigma-Aldrich, UK</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Micro BCA, Protein Assay Kit</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10249133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Odyssey&#160; blocking buffer</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td>P/N 927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg&#160;</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">ODYSSEY CL Infra-red imager</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;"></td>
			<td>Call for quotation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">iBlot 7-Minute Blotting System</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td style="width:119px;"></td>
			<td>This model is no longer in production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">InstantBlue Protein stain</td>
			<td style="width:255px;">Expedeon, UK</td>
			<td>ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Revitablot western blot stripping buffer&#160;</td>
			<td>Rockland Immunochemicals Inc.</td>
			<td>MB-085-0050</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to modern quantitative fluorescent western blotting with troubleshooting strategies

1970年代後半には、ウェスタンブロット、存在感、複雑な生物学的サンプル中の特定のタンパク質の相対存在量を評価するための技術の最初公に報告された使用を見た。それ以来、ウエスタンブロッティング法は、分子生物学実験的レパートリーの共通成分となっています。用語「ウェスタンブロット」を使用してのPubMedのざっと検索が22万公表原稿を超えて重要な年2014年までにこの技術を利用してきたことを示唆して、最後の10年間は​​、開発と相まってテクニカルイメージングの進歩を見てきました感度を改善し、線形検出のより大きな範囲が得られている敏感な蛍光標識。その結果、生物学者はこれまで以上に高い感度と精度を比較発現解析を行うことを可能にするウェスタンブロット（QFWB）ベースの今、真に定量化蛍光である。多くの &quot;最適化&quot;ウェスタンブロッティング方法論が存在し、異なる研究室で利用されている。これらは、しばしば微妙ですが、文書化されていない手続きの改正の要件に実施することは困難証明する。このプロトコルは、トラブルシューティングの戦略と完全に確立され、堅牢なQFWB方法の包括的な説明を提供します。

QFWB感度および検出の直線範囲は、従来のECL検出よりも大きいように、実効的な解釈を助ける正確なデータが収集されることを保証するために重要である制御手段の数は、存在する。まず、ポジティブ対照試料を含めることは、 図1に示すように第二に、転送の最適化は、膜にゲルから高および低分子量タンパク質の同等の動作を保証するために、図2に示したように、第三に、抗体の最適化は、特に、二次その最適化はしばしば見落とされているが、抗体は、関心対象のタンパク質の正しい解釈を妨害することができるバンディング非特異的生成することができる。 図3を参照してください。第四に、それはまた、タンパク質が検出不能に見えるが存在することが予想される場合、これは単に二RAISを用いて補正することができる二次抗体の問題であってもよい場合もある異なる種の宿主中で編図4を参照してください。第五に、総タンパク質標識および分析は、遍在内部参照標準3のために表されている伝統的な単一のタンパク質（群）の使用と比較してはるかに堅牢かつ定量化可能な方法である。これらの単一のタンパク質の多くは、示差的に神経変性疾患のモデルとの間、異なる組織サンプルにおいて発現されることが見出されており、発現の均一性は、同じ組織3内に変更することができる。標準誤差を示すために比較し、各サンプルに対して測定し、種々の分子量の範囲において、各レーンにおけるタンパク質負荷を定量化することによって、総タンパク質分析と組み合わされた場合、図6に示されているように、したがって、ローディング対照ゲルの製造は、サンプル負荷の均一性を確認する。重要なことに、これらのトラブルシューティングの手法とコントロールの全てが分析さtの感度および一貫性のみに有効であるオペレータ（ 図5）によって適用ools。最後に、この技術は、ストリッピング及び要因を含むが、長期貯蔵条件下での感受性の増大、減少、背景、デュアルカラー検出および膜安定性に限定されずにECLよりも柔軟性を有する膜の再プロービングに向いている。 図7を参照してください。

Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

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JoVE Journal

Biology

50.7K ビュー.  University of Edinburgh. The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

ビデオ: A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

西洋は、Invitrogen NuPage NOVEXビストリスミニゲルを用いたウェスタンブロット

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

生物学

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

遺伝子誘導の運命のマッピングへの実践的アプローチ：マークとトラックの細胞へのビジュアルガイドインビボでの</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

ウェスタンブロッティング：サンプル調製を検出する

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

ポリメラーゼ連鎖反応：基本的なプロトコルプラスのトラブルシューティングと最適化戦略

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

ウェスタンブロッティングにおける実用的で便利で信頼性の高い全タンパク質ローディング制御のためのV3染色不要のワークフロー

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Nonexpertsための系統学の実用ガイド

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

町の西部最速：クラシックウェスタンブロット分析に現代風にアレンジ

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

亜麻の花-DIP変換（<em&gt;アマ</em&gt;）高転換率のトランスジェニック子孫を生成するために、

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

多重化アプリケーションのための蛍光タンパク質との遺伝バーコーディング

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

内部陽性対照との定量的リコンビナーゼポリメラーゼ増幅アッセイの開発

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...