我々は彼らの技術的貢献のためにServane ContalとボリスUntereinerに感謝。我々は、FNR&quot;FUTOX&quot;プロジェクトのサポートを認める。

プロトコルは、その完成のための16日を要します。時間枠は、図1に詳述されています。 バッファーの調製のための詳細<ul><li>溶解バッファー（サンプルあたり3ML）の調製。 </li><li>洗浄バッファー（サンプル当たり50mLの）の準備。このバッファはあらかじめ冷却して保存-20℃冷凍庫でさらに分析されるまで、全体の手順の間に氷上で保存する。 </li><li>降水量のバッファー（サンプル当たり20mlの）の準備。全体の手順の実行中に必要とし、氷上に保持されるまで、このバッファは、冷凍庫で-20℃であらかじめ冷却して保存する必要があります。 </li><li>仕事が優先的に4℃のコールドチャンバー℃に行われるべきである</li></ul>真菌種プロテオーム解析のために使用する3つの菌株は、それらの形態学的特徴と翻訳伸長因子α- 1分析（オドネルら、1998）に基づいてフザリウムgraminearumに分類されたこれらの菌株はCRP -ガブリエルリップマンコレクションに所属し、維持し、で保存した - 80 ° C 15％グリセロール。 菌糸の準備菌類は25℃、光と暗期を交互に各12時間で4日間V8で培養した。培養物は、毒素誘導媒体100 mLを含む250ミリリットルの三角フラスコに接種するために使用されたこれらの培養物の滅菌ブレードとピースを使用して断片化された。菌糸体を10日後に回収し、滅菌フィルターで文化をフィルタリングすることにより、培地から分離した。菌糸体を培地残基と他の化合物を除去するために滅菌水で3回洗浄した。私たちは、その後すぐに液体窒素を添加することにより、細胞を凍結し、-80試料を保管℃にタンパク質抽出の一般的な説明タンパク質サンプルは、図3Aおよび3Bに記載された方法を用いて抽出した。 フザリウム属のサンプルは、液体窒素中で粉砕され、粉体を10mlテフロンチューブに回収した。次いで、試料を10分間煮沸、溶解緩衝液で30分インキュベートし、室温（15分間12000グラム）で2回の遠心分離した。上清を一晩沈殿バッファーを-20℃で収集し、インキュベートした。 45分間4℃3万グラムで遠心分離後、沈殿したタンパク質のペレットを冷アセトンを含むDTTで3回洗浄した。最後に、ペレットは、空気乾燥され、タンパク質は、pHを8に調整し、標識バッファーでresolubilizedれた。 30mlのサブサンプルは、総タンパク質の定量に除去し、残りの上清を蛋白質の電気泳動まで-20℃で保存した。 <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="図1" /> 図1の手順のタイムライン。 <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="図2" /> 図2。複数の演算子のサンプル処理のためのスキーム。 <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="図3a" border="0" /> 図3a。 <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="図3b" border="0" /> 図3bは。してください<a href="/files/ftp_upload/1690/1690fig3a.jpg">ここをクリックして</a>図3aの拡大バージョンを参照する、または<a href="/files/ftp_upload/1690/1690fig3b.jpg">ここで</a> 、図3bの大きいバージョンのために。 図3a - B蛋白質の抽出手順のフローチャート（1日目、B：二日目）。。 <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="図4" border="0" /> 図4。タンパク質抽出物の1D絵。タンパク質は、溶岩の紫で染色されたゲルの端に青色の完全移行するまで実行されました。してください<a href="/files/ftp_upload/1690/1690fig4.jpg">ここをクリックして</a>図4の拡大バージョンを参照すること。

<ol>
	<li>Bridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Extraction+from+Fungi.">Protein Extraction from Fungi.</a> Protein Purification Protocols. , 39-48 (1996).</li><li>Jiao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+carbon+sources+on+trichothecene+production+and+Tri+gene+expression+by+Fusarium+graminearum+in+liquid+culture.">Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture.</a> FEMS Microbiol Lett. 285, 212-219 (2008).</li><li>Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+filamentous+fungi.">Proteomics of filamentous fungi.</a> Trends in Biotechnology. 25, 395-400 (2007).</li><li>Milles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+proteomic+approach+to+monitor+protein+synthesis+in+mycotoxin+producing+moulds.">Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.</a> Mycotoxin Res. 23, 161-165 (2007).</li><li>Morrison, N., Field, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concept+of+sample+in+OMICS+technology.">Concept of sample in OMICS technology.</a> OMICS. 10, 127-137 (2006).</li><li>O'Donnell, K., Kistler, H. C., Cigelnik, E., Ploetz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+evolutionary+origins+of+the+fungus+causing+Panama+disease+of+banana:+Concordant+evidence+from+nuclear+and+mitochondrial+gene+genealogies.">Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies.</a> PNAS. 95, 2044-2049 (1998).</li><li>Paper, 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=Comparative+proteomics+of+extracellular+proteins+in+vitro+and+in+planta+from+the+pathogenic+fungus+Fusarium+graminearum.">Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.</a> Proteomics. 7, 3171-3183 (2007).</li><li>Pasquali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+in+science.+Protocol+videos:+the+implications+for+research+and+society.">Video in science. Protocol videos: the implications for research and society.</a> EMBO Rep. 8, 712-716 (2007).</li><li>Taylor, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+information+about+a+proteomics+experiment+(MIAPE).">The minimum information about a proteomics experiment (MIAPE).</a> Nat Biotechnol. 25, 887-893 (2007).</li><li>Taylor, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analyses+of+Fusarium+graminearum+grown+under+mycotoxin-inducing+conditions.">Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions.</a> Proteomics. 8, 2256-2265 (2008).</li></ol>

真菌生物学ドメイン内のプロテオミクスのアプローチの最近の関心は、このテクニックを（Kim ら 、2007年にレビュー）を使用して出版物の増加につながった。タンパク質抽出用のメソッドは、抽出手順の組み合わせに依存して、彼らはしばしば、ほとんどの方法論のセクションで説明しないと潜在的なトラブルシューティングの方法に対処する上で専門知識を必要としている。 他の&quot;オミクス&quot;アプローチ用として、サンプルおよびデータ処理のための基準を設定すると、信頼できる科学的情報を生成するために不可欠です。また、サンプルや操作の手順は適切に別の&quot;オミクス&quot;の実験間での共有の結果の解釈を可能にするために記述する必要があります。これは完全にゲノミクス、プロテオミクス、メタボロミクス、のクロスデータマイニングの可能性を活用するために不可欠になる... （モリソンら 、2006）。プロテオミクス実験に関する最低限の情報が起草され、ワー​​キンググループは、実験（ゲル電気泳動、質量分析、分子間相互作用、タンパク質修飾、プロテオミクスの情報、サンプルの処理）のあらゆる側面に取り組むために設立されました。現時点では定義されている情報は、サンプルの処理手順でダウンロード可能となります。 MIAPEガイドライン（Taylor ら、2007）の枠組みの中で標準化を高めることができるプロシージャを実装するためには、プロテオミクス研究の最終的な品質を決定する際のタンパク質の抽出手順の重要性を考えると、我々はサンプルを詳述したビデオプロトコルの使用を提案処理。実験のビデオの説明は&quot;オミクス&quot;の実験（パスカリ、2007）の優れた再現性をもたらすことができるサンプルの処理に関する情報の数を増やすために大幅に貢献するかもしれない。 確かに、研究室間再現性は、プロテオミクスの結果（http://www.fixingproteomics.org）の妥当性を保証するために基本的な要件です。別のインストルメンテーションとサンプルを操作する別の演算子：クロス実験の再現性は、2つの要因に影響されます。 記述されたプロトコルでは、我々は実行するために提案する生物学的データ（結果のすなわち技術的変化が考慮される）の信頼性を高めるために複数の演算子（図2およびビデオ）を含む複製。 タンパク質抽出のために説明する手順は、SDSと加熱に基づいています。これは、以前にタンパク質（大橋1996）の良好な純度と量を保証することが示された。沸騰と相まってSDSは、細胞壁、疎水性タンパク質とタンパク質の中止沈殿そのオリゴマーの形成を防止して溶解する。沸騰は、プロテアーゼの不活性化も可能です。同じ効果は、EDTA、PMSFおよび完全なミニプロテアーゼ阻害剤によっても生成されます。 DTTはタンパク質の可溶化を促進するタンパク質の間のジスルフィド架橋を削除します。 IEF前にSDSを除去するためには、タンパク質をアセトン/ TCA / DTTによって沈殿させる。さらに、アセトン/ TCA / DTT降水量は、これらの化合物が存在する場合、脂質、核酸、塩または/およびフェノール化合物などのいくつかの汚染物質を除去することができます。 SDSと同様に、これらの分子は、IEFの間に良好な移行を防ぐ。この沈殿に続いて、それはIEFと干渉する可能性としてTCAを除去するために、アセトン/ DTT、によって沈殿したタンパク質を洗浄する必要があります。 良好な試料調製が良い結果への鍵です。このため、パウダーフリーの手袋での作業と検査室の安全基準を尊重し、環境からの蛋白質の汚染を避けるためにも不可欠なことです。技術的な観点から、記述されたプロトコルの中で、蛋白質の十分な量と純度を得るために最も重要なステップは2つの段階があります。最初に、研削セルの完全な破壊は、タンパク質を解放するために基礎となる相と、第2、総タンパク質の精製、脂質、DNAとタンパク質の移行を妨げる可能性があります他の汚染物質の大部分の除去を包含する段階。

メディアとソリューション PDA：39 gのポテトデキストロース寒天（Difco社製）、1リットルの蒸留水。 V8寒天 ：200ミリリットルV8（キャンベル、米国）、2G炭酸カルシウム（シグマ）、16グラム寒天（DIFCO）、800 mlの蒸留水 毒素を誘導するメディア ：1G K 2 HPO 4、0.5グラムのKCl、0.5グラムのMgSO 4 7H 2 O、10mgのFeEDTA、2G Lグルタミン酸、蒸留水の1Lで10gショ糖。 溶解バッファー ： <table border="1"><tr><td></td><td> 100mLの</td><td>最終濃度</td><td></td></tr><tr><td>トリス- HCL PH8 </td><td> 5mLの</td><td> 50mmの</td><td> 1M </td></tr><tr><td> SDS </td><td> 2Gまたは10mLを</td><td> 2パーセント</td><td> 20パーセント</td></tr><tr><td> DTT </td><td> 500mLに</td><td> 10mMの</td><td> 2M </td></tr><tr><td> EDTA </td><td> 20mlの</td><td> 0.1mmの</td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200mLの</td><td></td><td></td></tr><tr><td>完全なミニプロテアーゼ阻害剤</td><td> 1錠</td><td></td><td></td></tr><tr><td>水</td><td> QSP </td><td></td><td></td></tr></table> 降水量のバッファ ： <table border="1"><tr><td></td><td> 100mLの</td><td>最終濃度</td></tr><tr><td> TCA </td><td> 20mlの</td><td> 20パーセント</td></tr><tr><td> DTT </td><td> 0.1mlを</td><td> 0.1パーセント</td></tr><tr><td>アセトン</td><td> QSP </td><td></td></tr></table> 洗浄バッファー： <table border="1"><tr><td></td><td> 100mLの</td><td>最終濃度</td></tr><tr><td> DTT </td><td> 0.1mlを</td><td> 0.1パーセント</td></tr><tr><td>アセトン</td><td> 100mLの</td><td></td></tr></table> ラベリングバッファー（-18℃で保存小分けにC）： <table border="1"><tr><td></td><td> 1mLの</td><td>最終濃度</td></tr><tr><td>尿素</td><td> 140 mgの</td><td> 7M </td></tr><tr><td>チオ尿素</td><td> 50mgの</td><td> 2M </td></tr><tr><td> CHAPS </td><td> 40 mgの</td><td> 4パーセント</td></tr><tr><td>トリス</td><td> 30μL </td><td> 30mMの</td></tr><tr><td>水</td><td> 1 mLの</td><td></td></tr></table>

toxin induction and protein extraction from fusarium spp. cultures for proteomic studies

fusariumの複数形は異なる毒素を生産することができる糸状菌である。このようなデオキシニバレノール、ニバレノール、T2、zearelenone、フザリン酸、moniliformin、などのフザリウムマイコトキシン..人間と動物の両方の健康に悪影響を与えると、一部は病原性の要因として考えられている。プロテオミクス研究は、毒素産生機構（Taylor ら 、2008）解読するだけでなく、fusariumの複数形で（紙ら 、2007、Houterman ら、2007）潜在的な病原性因子を同定するために有効であることが示された。それは実験、系統と実験室間のタンパク質発現に見られる真の違いに依存するためにプロテオミクス研究の間の比較のために信頼性の高い方法を確立することが基本になります。説明する手順は次の2つの方法でプロテオーム手続の標準化レベルの上昇に貢献すべきである。撮影プロトコルを正確に記述することができる詳細のレベルを高めるために使用されます。また、生物学的処理するために標準化された手順の可用性は、抽出作業の技術的な再現性の中でも、考慮に人的要因を考慮、データのより高いロバスト性を保障すべき複製します。 文化とタンパク質抽出のための2日間（図1）のための十四日：記述されるプロトコルは、その完成のための16日を要します。 簡単に言えば、 フザリウム株は 4日間固体培地上で増殖され、それらはその後、手動で10日間のために断片化と修正された毒素の誘導メディア（交通ら、2008。）に転送されます。菌糸体をミラクロス層を通して濾過することにより収集されます。粉砕は、コールドチャンバーで実行されます。異なる演算を実行抽出は考慮に入れ、技術の変化（図2）に起因するバイアスをとるために（n = 3）を複製します。抽出は、Taylorらに記載されてSDS / DTTバッファーに基づいていた。 （2008）わずかな修正で。総タンパク質の抽出は、アセトン/ TCA / DTTバッファー晩とDTT /アセトン洗浄（図3a、3b）を用いてタンパク質の沈殿処理が必要でした。タンパク質は、最終的にタンパク質標識バッファーでresolubilizedし定量した。抽出の結果は、2Dゲル（IEF / SDS - PAGE）に進む前に、1Dゲル（図4、SDS - PAGE）で可視化した。同じ手順は、他の成長しているメディアや他の糸状菌（マイルズら、2007）上のプロテオーム解析に適用することができます。

Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

真菌種のプロテオーム解析のためのタンパク質の抽出は、プロテオーム実験（MIAPE）ガイドラインに関する最小限の情報に応じて達成される標準化の高いレベルが必要です。私たちは、から毒素の誘導と蛋白質の抽出時に実験的なバイアスを最小限にするための手順が含まれているビデオプロトコルを提示<em&gt;フザリウム属。</em

から毒素誘導とタンパク質抽出フザリウム属属。</emプロテオーム研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

toxin-induction-protein-extraction-from-fusarium-spp-cultures-for

Nanyang Technological University

Research

JoVE Journal

Biology

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

16.9K ビュー.  Centre de Recherche Public-Gabriel Lippmann. 真菌種のプロテオーム解析のためのタンパク質の抽出は、プロテオーム実験（MIAPE）ガイドラインに関する最小限の情報に応じて達成される標準化の高いレベルが必要です。私たちは、から毒素の誘導と蛋白質の抽出時に実験的なバイアスを最小限にするための手順が含まれているビデオプロトコルを提示フザリウム属。

ビデオ: Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

RNAまたは特定の脳領域からのタンパク質抽出用のラット脳の微小解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

生物学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

タンパク質の電気泳動分離

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

インタビュー：タンパク質フォールディングと神経変性疾患の研究

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

ニューロンとグリアの開発研究ラットの脳の集計文化の調製

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

自動プロテオーム処理のためのデジタルマイクロ流体

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

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 ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

遺伝学的およびエピジェネティクス解析のためのパラフィン包埋材料からのDNA抽出

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

の性的開発と子嚢胞子放電フザリウムgraminearum種</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

から毒素誘導とタンパク質抽出フザリウム属属。文化