この作品は、クヌートとアリスウォレンバーグ財団からの補助金によって支えられている。ウプサラ、ストックホルム、インドの人間のタンパク質アトラスセンターの全体のスタッフは、ヒトタンパ​​ク質アトラスを生成するための努力のために承認されています。著者らは、撮影時に特にTMA制作の支援のための写真やエレーヌKarlbergの支援についてはフランクHammarとソフィー·グスタフソンに感謝したいと思います。

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarray:+a+new+technology+for+amplification+of+tissue+resources.">Tissue microarray: a new technology for amplification of tissue resources.</a> Cancer J. 7, 24 (2001).</li><li>Catchpoole, D., Mackie, N., McIver, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tape+transfer+sectioning+of+tissue+microarrays+introduces+nonspecific+immunohistochemical+staining+artifacts.">Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts.</a> Biotech. Histochem. , (2010).</li><li>UniProt. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 39, D214 (2011).</li><li>Uhlen, M., Oksvold, P., Fagerberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+knowledge-based+Human+Protein+Atlas.">Towards a knowledge-based Human Protein Atlas.</a> Nature Biotechnol. 28, 1248 (2010).</li><li>Leong, A. S., Leong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+in+immunohistology.">Standardization in immunohistology.</a> Method Mol. Cell Biol. 724, 37 (2011).</li></ol>

我々は、開示することは何もありません。

免疫組織化学は、はるかにTMASための最も一般的に使用されるアプリケーションです。大規模な患者のコホートから組織サンプルに基づいて、IHCとTMASの組み合わせは、いくつかの異なる設定で使用することができ、組織7 8および細胞9 10におけるタンパク質発現パターンの例：ハイスループットスクリーニング、バイオマーカー探索の研究11 12とより基本的な腫瘍の生物学異なる表現型/遺伝子型、分化段階、進行や転移13を含む研究では、。 TMASを使用する利点の1つは大規模コホートからその材料が貴重な生物学的材料を節約し、より再現性の実験を確実に両方、一度に分析することができます。また、TMA技術の使用は、試薬コストや研究室の処理時間を節約できます。 TMAのみが組織の限られた量が含まれているため、組織の不均一性が問題です。組織と対処すべき問題の種類に応じて、いくつかのサンプルが同じspecimeから必要になることがありますN。それは全体の組織切片を13 14の表現で高精度をもたらすことが示されているように腫瘍組織については、各標本二から四までのコアの使用をお勧めします。組織の組成に応じてパンチ（0.6ミリメートルから2ミリメートルまでの範囲）の直径を調整することができます。組織は、いくつかの異なる種類の細胞、構造および細胞外マトリックスの両方の複雑で構成されています。脂肪の可変量を含む、それぞれ異なる組織型の組成物は、血管、結合組織、軟骨などは、独立したコアをパンチングして収集するために必要な圧力に影響を与えます。それが意図した実験のために定義されている組織とTMAを生成する前に、任意の値ではありません材料にすべての手順を練習するに重要である。 1 TMAブロック内の異なる組織の様々なセクショニング時にブロックの構成は、高品質のセクションを取得する能力に影響を与える可能性がある。 CErtain組織、例えば皮膚、骨髄、脂肪、脳、乳房、セクションでは、水浴中で伸ばす方法差押質感の違いの影響を受けることにより困難にTMAブロックの切片とどのように異なる硬度レンダリングは、ブレード等により切断されしたがって、1 TMAは、該当するに似た機能などの脂質の豊富な組織を持つグループ組織することを推奨します。癌組織の大半はこの意味で同質であり、これは癌のTMASの切片が容易になります。組織の異種タイプのTMAを取り扱う際にはマイクロアレイコアを安定させるための代替的切断法を採用することができる。粘着テープの使用は、断面のTMAのコアと秋アウトの損失を避けるために役立ちます。テープ断面のコアの厚さに影響を与え、その後も、IHC染色15の結果できるので、粘着テープの使用は、慎重に使用すべきである。 9x8の人間のタンパク質アトラスのセットアップTMAテンプレート（マーカーを除く）コアのAReが使用されます。それは、セクションの最大数を取得し、ブロック全体で表されるすべての組織を維持するために重要である。試薬とスキャン時間を節約するために、2つのセクションは、9x8の最大のテンプレートを可能にする1スライドガラス上に配置されています。 タンパク質については、ユニバーサルタンパク質リソース16の主要なリポジトリでは、唯一の約66％がタンパク質レベルでの証拠を持っているのは約20.300件のヒトタンパク質のエントリが含まれています。また、タンパク質レベルでの存在の証拠があるのための遺伝子の大半は、希少またはタンパク質の機能と発現の分布に関する情報があります。今後の重要な課題は、分布パターンとヒト組織の様々なタイプのこれらの未知のタンパク質の相対量を分析することです。そのようなUNIPROT、ENSEMBL、PubMedから出版されたデータなどのデータベースからの情報は、免疫組織化学的染色パターンに対する抗体を使用している場合を確立するためのガイドとして使用することができます病棟未知のタンパク質は、目的の標的タンパク質の実際のタンパク質プロファイルを表しています。さらに、いくつかの他の検証戦略は、タンパク質配列、ウェスタンブロット、免疫蛍光法、免疫沈降やプルダウン実験の例では抗体の機能のために、抗体の機能を確保するために必要とされている。おそらく、抗体の機能を検証するための最も重要なツールでは、ペアの抗体を使用すること、すなわち2つの異なる抗体は、アッセイに固有の結果17を比較するために、同じ標的タンパク質上の別個の、重複しないエピトープに対して提起した。 コンパイルされた情報に基づいて、抗体はIHCの標準と人間のタンパク質アトラスプロジェクトの35.000抗体を介してテストおよび検証の経験に基づいてテストされ、滴定されています。タンパク質プロファイルを持つすべての遺伝子の20％以上の場合は、ペア抗体（同じタンパク質上の非重複エピトープに向かって2つ以上の抗体）からのデータは、最高の推定値を決定するために使用されています対応するタンパク質の発現パターンのメイト。ペアの抗体は、特定の免疫組織化学ベースのタンパク質発現パターンを検証するための究極の戦略を提供しています。基本的なスクリーニングに含まれている別の組織の多くは、交差反応のリスクを増加させるので、この戦略は、貴重なものです。それは特定の組織は、一般的にバックグラウンド染色例、マクロファージ、平滑筋、肝臓、腎臓及び肝細胞の遠位尿細管を表示する傾向があることにも留意すべきである。偽陰性または不適切なポジティブIHC染色パターンになることがアーカイブ材料から採取した組織のために時間をかけて組織の処理技術のバリエーションを含んで考慮すべきその他の問題。この現象は、大規模なセクション分析で検出されました。正および負の両方のコントロールが不可欠であり、可能な限り使用する必要があります。対応するTRの機能解析、強制発現細胞株および特定のノックダウンを含む、より深い研究のためのanscripts（siRNA技術）は、コントロールを作成するために使用することができます。ホルマリン固定、架橋などの様々な化学修飾、抗原18をマスキングシッフ塩基の加水分解を誘導するため、IHCで最適な結果を得るためには、それが抗原検索システムをテストし、使用することが重要です。 結論として、TMA技術およびIHCを採用した抗体ベースのプロテオミクスでは、大規模なタンパク質の発現データを生成するための強力な戦略です。人間のタンパク質アトラスプロジェクトは、ヒト正常組織および癌におけるヒトタンパ​​ク質発現のマップを作成するように設定されている。このプロジェクトの目的は、2015年までに包括的なヒトタンパ​​ク質アトラスの最初の草案を提示することである。正常な臓器や組織中のタンパク質プロファイルを提供することに加えて、この努力は、臨床バイオマーカー探索の努力を含む生物医学研究プロジェクトの出発点を提供します。究極の目標は、ヒトゲノム配列の上に次の情報レイヤーを追加することですwiを様々な疾患状態の根底にある生物学をより深く理解するために重要である、および新規診断·治療ツールを開発するための基礎を提供するでしょう。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>洗浄バッファーを</td><td>サーモフィッシャーサイエンティフィック</td><td> TA-999-TT </td></tr><tr><td>バッファpH6でのクエン酸</td><td>サーモフィッシャーサイエンティフィック</td><td> TA-250-Pm1X </td></tr><tr><td>抗体希釈液</td><td>サーモフィッシャーサイエンティフィック</td><td> TA-125-UD </td></tr><tr><td> UltraVision LP HRPポリマー</td><td>サーモフィッシャーサイエンティフィック</td><td> TL-125-HL </td></tr><tr><td> DAB基質を加えたシステム</td><td>サーモフィッシャーサイエンティフィック</td><td> TA-125-HDX </td></tr><tr><td>ウルトラVブロック</td><td>サーモフィッシャーサイエンティフィック</td><td> TA-125-UB </td></tr><tr><td>一次抗体エンハンサー</td><td>サーモフィッシャーサイエンティフィック</td><td> TL-125-PB </td></tr><tr><td>メイヤーズヘマトキシリン</td><td> HistolAB </td><td> 01820 </td></tr><tr><td> PERTEX </td><td> Histolab </td><td> 00871.0500 </td></tr><tr><td colspan="3"></td></tr><tr><td> 機器の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>マニュアル組織マイクロアレイヤー</td><td> Estigen、ビーチャーインストゥルメント</td><td> MTA-1 </td></tr><tr><td>滝ミクロトーム</td><td>サーモフィッシャーサイエンティフィック</td><td>マイクロモルHM 355S </td></tr><tr><td>自動化されたイメージスキャナ</td><td>アペリオ·テクノロジーズ</td><td> XT </td></tr><tr><td>自動化されたスライドの染色システム</td><td>サーモフィッシャーサイエンティフィック</td><td> Autostainer 480S-2D </td></tr><tr><td> deparafinizationと脱水のために自動化されたスライドの染色システム</td><td>ライカバイオシステムズ</td><td> Autostainer XL </td></tr><tr><td>自動化されたガラスcoverslipper </td><td>ライカビオスystems </td><td> CV5030 </td></tr><tr><td> Decloaking室</td><td>バイオケア·医療</td><td> DC2008INTEL </td></tr></tbody></table>

production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas

組織マイクロアレイ（TMA）技術は、複数の組織や細胞のハイスループット分析のための手段を提供します。技術は、グローバルな正常ヒト組織におけるタンパク質発現パターンの解析、癌および細胞株、ヒトのタンパク質アトラスプロジェクト内で使用されます。ここでは、単一の受信TMAブロックに微視的に選択された代表的な組織から取得した1 mmのコアのアセンブリを、紹介します。 TMAブロックの数とコアのサイズは約40 2ミリメートルのコアから0.6ミリメートルのコアを最大数百個に変化させることができる。 TMA技術を使用する利点は、そのデータの大量を急速に実験的な変動を避けるために、単一の免疫染色プロトコルを使用して得ることができる。重要なのは、希少な組織の限られた量が大きな患者コホート1の分析のために可能にする、必要とされる。約250の連続した​​切片（4μmの厚さ）TMAブロックからカットし、免疫組織化学的stainiに使用することができます250種類の抗体に特定のタンパク質の発現パターンを決定するためにNG。人間のタンパク質アトラスプロジェクトでは、抗体は、すべてのヒトタンパ​​ク質に向かって生成され、ヒトの癌の20の最も一般的な形式を表す216別の患者から、144の個人および癌組織の両方から正常なヒトの組織で対応するタンパク質のプロファイルを取得するために使用されています。免疫組織化学的にスライドガラス上に染色されたTMAのセクションは、病理医は免疫組織化学の結果を解釈し、注釈を付けることができ、そこから高解像度の画像を作成するためにスキャンされます。一緒に対応する病理学ベースの注釈データを持つイメージは、人間のタンパク質アトラスポータル（を通じて研究コミュニティのための公に利用できるようになります<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a> ）（ 図1）3。ヒトタンパ​​ク質アトラスは、人体内のタンパク質の分布と相対量を示す地図を提供しています。現在のバージョンシオンは、ヒトのゲノムによってコードされるすべてのタンパク質以上の61パーセントに相当する12.238ユ​​ニークな蛋白質のタンパク質発現データと1,100万件以上のイメージが含まれています。

Watch this Scientific Journal Video about Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas at JoVE.com

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

組織マイクロアレイは、組織の多数からの同時情報を得るために効率的な方法が可能になります。組織の代表的な部品は、単一のパラフィンブロックに組み立てられます。ブロックからのセクションは、タンパク質の発現パターンの免疫組織化学および分析のために使用されます。デジタルスキャンは、データの分布に対応する画像を生成する。

ヒトタンパ​​ク質アトラス内の組織マイクロアレイ、免疫組織化学染色とデジタル化の生産

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

production-tissue-microarrays-immunohistochemistry-staining

Nanyang Technological University

Research

JoVE Journal

Biology

55.9K ビュー.  Uppsala University. 組織マイクロアレイは、組織の多数からの同時情報を得るために効率的な方法が可能になります。組織の代表的な部品は、単一のパラフィンブロックに組み立てられます。ブロックからのセクションは、タンパク質の発現パターンの免疫組織化学および分析のために使用されます。デジタルスキャンは、データの分布に対応する画像を生成する。

ビデオ: Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

組織サンプルのγH2AX病巣の定量

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

微小環境マイクロアレイの作製と利用（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

全取り付ける染色を用いた 3 D 白色脂肪組織構造の可視化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超構造保存細胞における膜蛋白質局在化の電子顕微鏡分析のためのクライオAPEX法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

ヒトタンパク質アトラス内の組織マイクロアレイ、免疫組織化学染色とデジタル化の生産

要約

さらに動画を探す

この動画の章

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

組織サンプルのγH2AX病巣の定量

微小環境マイクロアレイの作製と利用（MEArrays）

EMBLでMultiBacタンパク質複合体生産プラットフォーム

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで

全取り付ける染色を用いた 3 D 白色脂肪組織構造の可視化

超構造保存細胞における膜蛋白質局在化の電子顕微鏡分析のためのクライオAPEX法

ヒトタンパ​​ク質アトラス内の組織マイクロアレイ、免疫組織化学染色とデジタル化の生産

要約

さらに動画を探す

この動画の章

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

組織サンプルのγH2AX病巣の定量

微小環境マイクロアレイの作製と利用（MEArrays）

EMBLでMultiBacタンパク質複合体生産プラットフォーム

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで

全取り付ける染色を用いた 3 D 白色脂肪組織構造の可視化

超構造保存細胞における膜蛋白質局在化の電子顕微鏡分析のためのクライオAPEX法

ヒトタンパク質アトラス内の組織マイクロアレイ、免疫組織化学染色とデジタル化の生産