イタリアの保健省はこの研究を支持した（RC 2013-BIO 15）。彼女の優れた技術的支援のためにBarbara Micheliに感謝します。

このプロトコルでは、正常な心エコー検査のパラメーターにかかわらず、技術的または機能的理由のために、臓器移植から除外された多臓器ドナーからの多臓器外植片（4〜12時間の冷虚血時間、平均6±2時間）の間にヒト心臓を収集する。彼らは、大動脈弁および肺動脈弁の銀行業務のために、ミラノの心血管組織バンクMonzino Cardiologic Centre（ミラノ、イタリア）に送られる。僧帽弁後小葉は臨床目的では使用されないので、ドナーの親族からインフォームドコンセントが得られた後、大動脈弁および肺動脈弁の隔離中に収集される。移植および研究のための組織は、親の同意の後にのみ収集される。倫理委員会のガイドラインに従い、人間の臨床使用（ すなわち、微生物学的、機能的および血清学的問題）に適していない場合にのみ、同意書上で心臓組織の使用を認可する（またはしない）モンティーノ心臓病センター。 1.僧帽弁の準備<ol><li>臓器の外植後（冷虚血時間は4〜12時間）、できるだけ早くヒト僧帽弁を採取する。 </li><li>クリーンルームでは、冷たい（4℃）溶液（ すなわち、生理食塩水または平衡培地EurocollinsまたはWisconsin）を含む輸送用バッグから心臓を取り除く。それをバケツに入れて、バルブの準備を進めるために、バイオセーフティキャビネット（バイオハザード気流、クラスA、グッドマニュファクチャリングプラクティス（GMP）分類）に入れてください。 </li><li>キャビネット内の滅菌使い捨てドレープに心臓を置きます。滅菌使い捨てメスを使用して、心臓をその主軸に対して垂直に、心尖部から約4cm離れた左右の心室のレベルで完全に切断する。 </li><li>左心房の屋根を表示するために、上行大動脈と肺動脈を動かす。 </li><li>滅菌オートクレーブ可能な鉗子とピックを使用して、左耳の周りを僧帽弁を目に見えるようにし、大きな僧帽弁尖（前側）と小さい僧帽弁葉（後側）を識別することを可能にする。 注：前側および後側の交連は、前尖および後部の境界を画定する。 </li><li>滅菌オートクレーブ可能なはさみおよび非外傷性の鉗子を使用して、左心房および僧帽弁全体の周囲の心室壁の厚さを切開する。 </li><li>大動脈弁の連続性を確認する。 注：左心室には、僧帽弁全体と腱索が含まれています。 </li><li>前僧帽弁小葉を後僧帽弁小葉から分離し、後尖を脳室の挿入に沿って切断する（交連）。 </li><li> 生理食塩水中の後弁膜を洗浄する。リーフレットを小片（&lt;1 cm 2 ）に切断し、個々にアルミホイルで包んでください。液体窒素でそれらをスナップフリーズする。 注意：液体窒素を使用する場合は、組織の安全手順に従ってください。 <ol><li>手順の最後に、70％イソプロピルアルコール溶液と6％過酸化水素水でキャビネットのテーブルを衛生化してください。 </li></ol></li></ol> 2.タンパク質抽出<ol><li>鉗子を使用して、液体窒素に保存されたサンプルをピックアップし、直ちにアルミ箔に包みながらドライアイス上に置きます。移送中にサンプルを融解させないでください。 </li><li>粉砕する前に、液体窒素（約500mL）を入れたDewarフラスコに入れることによって、粉砕システム（ 例えば、 CryoGrinder）の磁器/ジルコニウムモルタルおよび乳棒を試料とともに冷やす。 注意：液体窒素を使用するときは、組織の安全手順に従ってください。 </li><li>ドライアイスを入れたポリスチレンボックスに乳鉢と乳棒を入れます。サンプルをアルミホイルから取り出し、モルタルに入れます。 </li><li> グラインドt彼は乳棒を回転させるためにドライバーを使用して、モルタルに対して大きな乳棒で15-20回サンプリングします。粉砕プロセスの間、予め冷却したスパチュラの先端と試料を混合する。 <ol><li>小さな乳棒で繰り返します。 </li></ol></li><li> チューブを反転させ、モルタルの上に置き、それらを一緒に反転させてサンプルをチューブに移動させることによって、前に計量したチューブ（ 例えば、 15mL遠心チューブ）に地上サンプルを移す。予備冷却されたスパチュラを使用して、モルタルからすべての材料を回収する。 <ol><li>移送中のサンプル解凍を避けるために、サンプルをドライアイス上に置いてください。 </li></ol></li><li>サンプルの正味重量を計算する。 </li><li>各サンプルの後に乳鉢と乳棒をきれいにし、オートクレーブまたは200°Cで2時間加熱することによってそれらを除染する。 </li><li>粉末サンプルを遠心分離管からホモジナイザーのガラス管に転倒させて移す。 </li><li>ろ過された尿素バフを加える10mgの粉末組織につき200μLの尿素緩衝液をガラス管に添加した（8M尿素、2Mチオ尿素、4％w / v CHAPS、20mMトリス、および55mMジチオトレイトール）。 注記：遠心チューブに残っている残留粉末サンプルは、計算された量の尿素バッファーの一部を使用して回収することができます。 </li><li>ホウケイ酸ガラス乳鉢およびポリテトラフルオロエチレン（PTFE）乳棒を備えた撹拌機を用いて試料を均質化する。サンプルをゆっくりと捻るような動き（1,500 rpm）で10回押します。 </li><li>上清を回収し、それを清潔な1.7mL遠心管に移す。新たな尿素緩衝液で残りの試料を再度抽出し、最初の抽出の間に使用した容量の半分を加える。 </li><li>手順2.10を繰り返します。 </li><li>上清を回収し、それをステップ2.11の上清と合わせる。合わせた上清をチューブローテーター上に30分間置く。 </li><li> 13,000 xgおよび4℃で30分間チューブを遠心分離する。 </li><li>上清を回収するBradfordタンパク質アッセイを用いて、製造業者の指示に従ってタンパク質濃度を測定する。サンプルを使用するまで-80℃で保存します。 </li></ol>

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著者は何も開示することはない。

このプロトコールの1つの重要なステップは、試料を凍結させ、粉砕システムを冷却するために液体窒素を使用することである。液体窒素の使用は生物分解を防ぎ、パウダー化を効率的に行うことができますが、安全な取り扱いのためには特別な訓練が必要です。 このプロトコールでは、標準的なモルタルおよび乳棒から小さなサンプルを回収することが困難であるため?...

最近の証拠は、mRNA合成後に生じる多くの調節機構の役割の理解を変化させている。実際、翻訳、転写後およびタンパク質分解プロセスは、タンパク質の存在量および機能を調節することができる。転写産物レベルがタンパク質存在量の主な決定因子であると仮定して、mRNA濃度が対応するタンパク質のプロキシであると言うドグマは部分的に改訂されている。細胞1,2内のタンパク質を調節するために起こる。 さらに、タンパク質は最終的に細胞の機能を決定し、したがって自己分泌、傍分泌および内分泌因子に応答して動的変化を起こすことができるその表現型を指示する。血液媒介メディエーター;温度;薬物治療;病気が発症する。このように、タンパク質レベルに焦点を当てた発現解析は、プロテオームの特徴を明らかにするとともに、疾患病因の一部として起こる重大な変化を解明するのに有用である3 。 したがって、既存の技術的課題にもかかわらず、プロテオミクスが健康状態や病状を明らかにする機会が大いにあります。プロテオミクスが貢献できる研究の特に有望な分野には、以下のものが含まれる：任意のレベル（ すなわち、細胞全体または組織、細胞内コンパートメントおよび生物学的液体）におけるタンパク質発現の変化の同定;疾患の診断および予後に有用な新規なバイオマーカーの同定、検証および検証;薬効と毒性の評価だけでなく、治療目的でも使用できる新しいタンパク質標的の同定4 。 複雑さを捉えるプロテオームは技術的な課題である。現在のプロテオミクスツールは、変化したタンパク質レベルの同定、定量化、および検証のための大規模で高スループットの分析を行う機会を提供する。さらに、最も豊富なタンパク質によって引き起こされる干渉を回避することを目的とする分画および濃縮技術の導入は、最も豊富でないタンパク質を含むことによってタンパク質同定を改善した。最後に、プロテオミクスは、タンパク質機能の重要なモジュレーターとして徐々に出現する翻訳後修飾の分析によって補完されてきた。 しかし、分析中の生物標本における試料調製およびタンパク質回収は、プロテオミクスのワークフローにおける制限的なステップのままであり、潜在的な落とし穴の可能性を高めます5 。実際、最適化されなければならない分子生物学技術の大部分において、最初のステップは、組織ホモジナイズイオンおよび細胞溶解、特に増幅方法が存在しない低濃度のタンパク質の分析中に起こる。さらに、タンパク質の化学的性質は、それら自身の回復に影響を及ぼす可能性がある。例えば、高度に疎水性のタンパク質の分析は、等電点電気泳動の間に容易に沈殿するため、膜貫通タンパク質はほとんど不溶性であるため、非常に難しい（参考文献5で検討されている）。さらに、組織組成の変動性は、普遍的な抽出方法を開発する上で重要な障壁となる。最後に、臨床検体のほとんどすべてが限られているため、最小限の検体量で最大の回収率と再現性でタンパク質を調製することが不可欠です。 この研究は、プロテオーム分析のための非常に困難なサンプルである、正常ヒト心臓僧帽弁からのタンパク質抽出のための最適化されたプロトコールを記載する。正常な僧帽弁は、心臓の左心房と左心室との間に横たわるレックス構造（ 図1 ）。心房から心室への血流を制御し、逆流を防止し、全身への適正な酸素供給を保証し、それによって十分な心拍出量を維持するのに重要な役割を果たす。しかし、それはしばしば細胞性が低く、主に細胞外マトリックス中の成分が少ない「不活性」組織であると考えられている。これは、正常な状態では、常在性の弁間質細胞（VIC）が低いタンパク質生合成速度で静止表現型を示すためである7 。 しかしながら、病理学的状態では、海綿体におけるVICの数が増加し、それらのタンパク質合成が他の機能的および表現型の変化と共に活性化されることが実証されている8 。したがって、驚くべきことではない文献は、増加した数の活性化VICが同定されたタンパク質の比較的高い数を説明し得る病理学的僧帽弁9,10の分析に焦点を当てている。 結論として、本プロトコールは、僧帽弁タンパク質成分の研究を通して、僧帽弁疾患に関与する病原機構の理解を発展させるのに役立つ可能性がある。事実、根底にある病理学的プロセスのより深い理解は、現在の介入適応症が血行力学的考察に主に基づいている弁疾患の臨床管理を改善するのに役立つ可能性がある。

<table><tbody><tr><td>Saline solution</td><td></td><td></td><td>0.9 % NaCl</td></tr><tr><td>Eurocollins A</td><td>SALF</td><td>30874046</td><td>Balanced organ&#039;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td></tr><tr><td>Eurocollins B</td><td>SALF</td><td>30874022</td><td>Balanced organ&#039;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td></tr><tr><td>Wisconsin</td><td>Bridge life</td><td>RM/N 4081</td><td>Balanced organ&#039;s transport medium</td></tr><tr><td>Biohazard vertical flow air</td><td>Burdinola</td><td></td><td>Class A GMP classification</td></tr><tr><td>Dewar Flask</td><td>Thermo Scientific</td><td>Nalgene 4150-1000</td><td></td></tr><tr><td>Cryogrinder system</td><td>OPS diagnostics</td><td>CG 08-01</td><td>Grinder system containing mortars, pestles and screwdriver</td></tr><tr><td>Stainless steel forceps</td><td></td><td></td><td></td></tr><tr><td>Stainless steel spatula</td><td></td><td></td><td></td></tr><tr><td>Disposable sterile scalpel</td><td>Medisafe</td><td>MS-10</td><td></td></tr><tr><td>Stainless steel scissors</td><td></td><td></td><td>Autoclavable</td></tr><tr><td>Stainless steel picks</td><td></td><td></td><td>Autoclavable</td></tr><tr><td>Disposable sterile drap</td><td>Mon&amp;amp;Tex</td><td>3.307.08</td><td></td></tr><tr><td>Sterilizing solution with isopropyl alcohol</td><td></td><td></td><td>70% isopropyl alcohol</td></tr><tr><td>Sterilizing solution with hydrogen peroxide</td><td></td><td></td><td>6% hydrogen peroxide</td></tr><tr><td>Micropipette, 1 mL, with tips</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>VWR international</td><td>9278</td><td></td></tr><tr><td>1.7 mL centrifuge tubes</td><td>VWR international</td><td>PIER90410</td><td></td></tr><tr><td>Urea buffer</td><td></td><td></td><td>8 M urea, 2 M thiourea, 4 % w/v CHAPS, 20 mM Trizma, 55 mM Dithiotreitol</td></tr><tr><td>Urea</td><td>Sigma aldrich</td><td>U6504-1KG</td><td>To be used for Urea buffer</td></tr><tr><td>Thiourea</td><td>Sigma aldrich</td><td>T8656</td><td>To be used for Urea buffer</td></tr><tr><td>CHAPS</td><td>Sigma aldrich</td><td>C3023-5GR</td><td>To be used for Urea buffer</td></tr><tr><td>Dithiotreitol</td><td>Sigma aldrich</td><td>D0632-5G</td><td>To be used for Urea buffer</td></tr><tr><td>Syringe 50 mL</td><td>PIC</td><td></td><td>To be used to filter Urea buffer</td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Millipore</td><td>SLGP033RB</td><td>To be used to filter Urea buffer</td></tr><tr><td>PFTE Pestle, 2 mL</td><td>Kartell</td><td>6302</td><td>Part of Potter-Elvehjem homogenizer</td></tr><tr><td>Borosilicate glass mortar</td><td>Kartell</td><td>6102</td><td>Part of Potter-Elvehjem homogenizer</td></tr><tr><td>Stirrer</td><td>VELP scientifica</td><td>Stirrer DLH</td><td>To be used for homogenization by Potter-Elvehjem</td></tr><tr><td>Bradford Protein assay</td><td>Bio-Rad laboratories</td><td>5000006</td><td></td></tr><tr><td>Tube rotator</td><td>Pbi International</td><td>F205</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Polystyrene box</td><td></td><td></td><td></td></tr><tr><td>Dry ice</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td>For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g</td></tr><tr><td>Freezer -80&amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>Precision balance</td><td></td><td></td><td></td></tr><tr><td>Autoclave</td><td></td><td></td><td>For sterilization</td></tr><tr><td>Cryogenic gloves for liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Professional forced ventilation and natural air convection oven</td><td></td><td></td><td>For sterilization</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma aldrich</td><td>P8340-5ML</td><td>100X solution</td></tr><tr><td>ProteoExtract Protein Precipitation Kit</td><td>Calbiochem</td><td>539180</td><td></td></tr><tr><td>RapiGest</td><td>Waters</td><td>186001861</td><td></td></tr><tr><td>Cytoscape</td><td>www.cytoscape.org</td><td>version 2.7</td><td>Software platform for Gene Ontology analysis</td></tr><tr><td>BiNGO</td><td>http://apps.cytoscape.org/apps/bingo</td><td>version 3.0.3</td><td>Plugin for Gene ontology analysis</td></tr><tr><td>AlphaB Crystallin/CRYAB Antibody</td><td>Novus Biologicals</td><td>NBP1-97494</td><td>Mouse monoclonal antibody against CryAB</td></tr><tr><td>Septin-11 Antibody</td><td>Novus Biologicals</td><td>NBP1-83824</td><td>Rabbit polyclonal antibody against septin-11</td></tr><tr><td>FHL1 Antibody</td><td>Novus Biologicals</td><td>NBP-188745</td><td>Rabbit polyclonal antibody against FHL-1</td></tr><tr><td>Dermatopontin Antibody</td><td>Novus Biologicals</td><td>NB110-68135</td><td>Rabbit polyclonal antibody against dermatopontin</td></tr><tr><td>Goat Anti mouse IgG HRP</td><td>Sigma aldrich</td><td>A4416-0.5ML</td><td>Secondary antibody for immunoblotting</td></tr><tr><td>Goat Anti rabbit IgG HRP</td><td>Bio-Rad laboratories</td><td>170-5046</td><td>Secondary antibody for immunoblotting</td></tr></tbody></table>

optimized protocol for the extraction of proteins from the human mitral valve

細胞のプロテオームの解析は、複雑な生物システムに存在するタンパク質の大規模な同定と定量を可能にする技術の開発による疾患の根底にある分子メカニズムを解明するのに役立ちます。プロテオームアプローチから得られた知識は、疾病の根底にある病原性メカニズムをより良く理解し、新規の診断および予後の疾患マーカーの同定を可能にする。しかし、心臓僧帽弁は、プロテオグリカンおよびコラーゲン富化細胞外マトリックスの細胞性が低いため、プロテオーム分析のための非常に困難なサンプルである。これは、全体的なプロテオーム分析のためにタンパク質を抽出することを困難にする。この研究は、定量的プロテオミクスおよびイムノブロッティングのような後続のタンパク質分析と互換性のあるプロトコールを記載する。これにより、データの関連性を考慮することができますgタンパク質の発現を、定量的mRNA発現および非定量的免疫組織化学的分析に関するデータと比較した。実際、これらのアプローチは、一緒に実施されると、mRNAから翻訳後タンパク質改変まで、疾患の根底にある分子メカニズムのより包括的な理解につながるであろう。したがって、この方法は、心臓弁の生理病理学の研究に関心のある研究者に関連する可能性がある。

ウレア緩衝液中のタンパク質の抽出および溶解は、等電点電気泳動（2次元電気泳動（2-DE） 11および液相等電点電気泳動（IEF） 12 ）に基づくプロテオーム法およびLaemmli緩衝液13での希釈後のイムノブロッティングプロテアーゼ阻害剤カクテル14を含有する。 <p class="jove_content" fo:keep-together.within-...

Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

ヒト僧帽弁のタンパク質組成は、未分化であり、その分析は細胞密度が低く、したがって低タンパク質生合成によって複雑であるため、まだ未知である。この研究は、僧帽弁プロテオームの分析のためにタンパク質を効率的に抽出するためのプロトコールを提供する。

ヒト僧帽弁からのタンパク質抽出のための最適化プロトコル

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

optimized-protocol-for-extraction-proteins-from-human-mitral

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

12.8K ビュー.  Centro Cardiologico Monzino IRCCS. ヒト僧帽弁のタンパク質組成は、未分化であり、その分析は細胞密度が低く、したがって低タンパク質生合成によって複雑であるため、まだ未知である。この研究は、僧帽弁プロテオームの分析のためにタンパク質を効率的に抽出するためのプロトコールを提供する。 

ビデオ: Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled哺乳動物細胞からのタンパク質複合体の親和性のキャプチャ

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

生化学

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 細胞から Mitoribosome の急速な隔離

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

動的プロテオミクスおよび miRNA 蛍光灌流後分離されたマウス心臓ポリソームの分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

ミトコンドリアの呼吸鎖 Supercomplexes の分離と分析のためのハイブリッドクリア/ブルーネイティブ電気泳動

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

骨格筋ミトコンドリアのタンパク質輸入能の測定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

<meta charset="utf8"></head><body>高品質の生体エネルギー研究のためのげっ歯類からのミトコンドリア単離

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>著者スポットライト:心不全と生体エネルギー学におけるミトコンドリアリン酸カルシウムの役割の解明

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

げっ歯類の心臓組織からの堅牢なミトコンドリア単離 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

に基づいて、タンパク質 - タンパク質相互作用の同定のためのプロトコル<sup&gt; 15</sup&gt; Nの代謝標識、免疫沈降法、定量的質量分析法と親和性調節

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

生物学

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

途上胚性マウス心臓弁地域からのタンパク質の単離

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

ヒト僧帽弁からのタンパク質抽出のための最適化プロトコル

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ミトコンドリアの呼吸鎖 Supercomplexes の分離と分析のためのハイブリッドクリア/ブルーネイティブ電気泳動

骨格筋ミトコンドリアのタンパク質輸入能の測定

げっ歯類の心臓組織からの堅牢なミトコンドリア単離

げっ歯類の心臓組織からの堅牢なミトコンドリア単離

げっ歯類の心臓組織からの堅牢なミトコンドリア単離

に基づいて、タンパク質 - タンパク質相互作用の同定のためのプロトコル

途上胚性マウス心臓弁地域からのタンパク質の単離