我々は科学的なサポートのためにマイケルReth、ヘルマンSchägger、とマルガリータカマチョ-カルバハルに感謝する。この作品は、ドイツ学術振興協会（DFG）からのBIOSではによって、Bundesministerium福二BildungとForschung（BMBF）からFORSYSによって賄われ、ドイツ連邦政府と州政府のエクセレンスイニシアチブによって部分的にサポートされていました（GSC - 4、シュペーマン大学院）。

<ol>
	<li>Arnold, I., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+F1F0-ATP+synthase+exists+as+a+dimer:+identification+of+three+dimer-specific+subunits.">Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.</a> EMBO J. 17 (24), 7170-7178 (1998).</li><li>Camacho-Carvajal, M. M., Wollscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+Blue+native/SDS+gel+electrophoresis+of+multi-protein+complexes+from+whole+cellular+lysates:+a+proteomics+approach.">Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.</a> Mol. Cell. Proteomics. 3 (2), 176-182 (2004).</li><li>Heiss, K., Junkes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subproteomic+analysis+of+metal-interacting+proteins+in+human+B+cells.">Subproteomic analysis of metal-interacting proteins in human B cells.</a> Proteomics. 5 (14), 3614-3622 (2005).</li><li>Sali, A., Glaeser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+words+to+literature+in+structural+proteomics.">From words to literature in structural proteomics.</a> Nature. 422, 216-225 (2003).</li><li>Schägger, H., Cramer, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217 (2), 220-230 (1994).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal. Biochem. 199 (2), 223-231 (1991).</li><li>Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation+of+protein+complexes+may+lead+to+aggregation+as+well+as+to+loss+of+subunits+as+revealed+by+Blue+Native+PAGE.">Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.</a> J Immunol Methods. 252 (1-2), 171-174 (2001).</li><li>Schamel, W. W., Arechaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+multivalent+and+monovalent+TCRs+explains+high+sensitivity+and+wide+range+of+response.">Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response.</a> J. Exp. Med. 202, 493-503 (2005).</li><li>Schamel, W. W., Reth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+and+oligomeric+complexes+of+the+B+cell+antigen+receptor.">Monomeric and oligomeric complexes of the B cell antigen receptor.</a> Immunity. 13 (1), 5-14 (2000).</li><li>Swamy, M., Minguet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+native+antibody-based+mobility-shift+technique+(NAMOS-assay)+to+determine+the+stoichiometry+of+multiprotein+complexes.">A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes.</a> J Immunol Methods. 324 (1-2), 74-83 (2007).</li><li>Swamy, M., Siegers, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+the+identification+and+analysis+of+multiprotein+complexes.">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.</a> Sci STKE. 2006 (345), 4-4 (2006).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8, 3974-3990 (2008).</li></ol>

利害の衝突は宣言されません。

本研究では、BN - PAGEによるMPCの分析について説明します。 2D的なアプローチは、ネイティブ条件下でまず個別のMPCに使用され、その後、さらに二次元目SDS - PAGEによって彼らの個々のコンポーネントにそれらを再分割する。 サンプルは、細胞ライセートから調製されています。多くのMPCの可溶化のために、適切な洗剤は、タンパク質複合体の構造は保存されますが、必要とされる。ここでは、0.1％トリトンX - 100を使用してください。しかし、最適な洗剤とその適切な濃度は、すべてのMPCのために経験的に決定する必要があります。トリトンX - 100の場合には、例えば、それは低い界面活性剤濃度は、F 1 F 0 - ATPaseの複合体（アーノルド、ファイファーら 1998）の二量体形態の識別を可能にすることが報告されている。高いトリトンX - 100濃度は、しかし、二量体の解離と単量体F 1 F 0 - ATPaseの複合体の対応する増加につながる。高濃度のまたは別の洗剤の使用量がINジギトニンの結果を呼ばれるのに対し、これは私たちのかつての研究の一つの線の上に、我々は、のBrij 96の低濃度で抽出するときに多価のT細胞受容体複合体（TCR）が保持されることを示していた単量体のTCRの抽出（Schamel、Arechaga ら、2005）。テストすることができる一般的に使用される洗剤は、ジギトニン（0.5〜1％）、トリトンX - 100（0.1〜0.5％）、のBrij 96（0.1〜0.5％）、またはdodecylmaltosideを（0.1〜0.5％）含まれています。これらの試薬は、MPCの安定のために最良になりがちな非イオン性界面活性剤、です。 SDSやその他の強力な洗剤との接触は（カマチョ-カルバハル、Wollscheid ら 2004）は避けるべきであることに注意してください。 溶解液の透析は、BN -ゲル（。カマチョ-カルバハル、Wollscheid ら 2004）でMPCの分離を達成するために必要とされ、（ハイス、Junkes ら、2005。）。それは、塩濃度または低分子量不純物の除去の調整は、高解像度のために重要であると思われる。また、免疫精製し、後で抗体から溶出されている膜調製物とMPCは、、（スワミー、Siegers ら 2006）BN - PAGEに適していることは注目に値する。どちらの場合も、サンプルは膜の溶解または溶出がBN -溶解緩衝液中で実施されている場合、BN - PAGEの分離のために透析する必要はありません。 BN - PAGEによるタンパク質の分離のために、色素クーマシーブルーは、タンパク質に非特異的に結合し、負電荷でそれらをカバーする、必要とされる。これにより、クマシーブルーでは、中性pH（SchäggerとフォンJagow 1991）で、陰極に向かって、タンパク質の電気泳動移動度を可能にします;（Schägger、クラマーら1994。）。さらに、クマシーブルーでは電気泳動中にスタッキングゲルにタンパク質の凝集を防ぎます。 BN - PAGEの場合は、クマシーG250は、クマシーブルーR250またはコロイドクマシーブルースの代わりに使用する必要があります。 BN -ゲルを実行する前に、それはゲルの割合は、関心のMPCの予想サイズに収まるようにするために必要です。さまざまなグラデーションと、適切なバッファを持つBN -ゲルはインビトロジェン（NativePAGE NOVEXビス - トリスゲルシステム）から市販されているプレキャスト。しかし、BN -ゲルはまたpersistalticポンプと一緒に勾配のミキサーを用いて調製することができる。無傷の勾配を保証するために、液体が注ぐ中に絶えず流れる必要があり、気泡は避けてください。オーバーロードは、電気泳動プロセス中にタンパク質沈殿する可能性があるため私たちは、ゲル上の異なるサンプルの希釈のロードをお勧めします。さらに、BN -ゲルは4℃で実行する必要があります° Cは、タンパク質の分解を防止するとMPCがそのまま保持する。 MPCはのBN - PAGE、可視化は、クマシーブリリアントブルー染色、銀染色やイムノブロッティングによって達成することができます後。タンパク質バンドはクーマシーまたは銀染色により可視化質量分析法（カマチョ-カルバハル、Wollscheid ら 2004）によるさらなる分析に適しています。イムノブロットの場合には、関心のMPCのための最適な転送条件は、経験的に決定する必要があります。クマシーブルーでもBN -ゲルからブロッティング中に転送されていることに注意してください。したがって、ゲル、膜は青色を発揮するのに対し、転送が成功すると無色になります。さらに、SDS - PAGE後の検出のために働くしないすべての一次抗体は、、BN - PAGEによりイムノブロットに適用可能であることを言及することが重要です。それは彼らのエピトープは、タンパク質の天然構造に隠されているため、抗体は興味のMPCを認識しないことが起こり得ます。この問題を克服するために、それは1x SDSサンプルバッファーでまもなくゲルを煮沸することにより前の転送へのBN -ゲル内のタンパク質を変性させることが可能です。 この例では、我々は、タンパク質バンドの検出に直接BN -ゲルを衝撃はなかった。その代わりに、我々は、第2 dimensiによってBN - PAGEで区切られたライセートを分けるSDS - PAGE上で。二次元SDS -ゲルでは、単量体蛋白質は第1および第2の次元（カマチョ-カルバハル、Wollscheid ら 2004）の線形ゲルの濃度勾配ゲルによる双曲線角内に移行する。彼らはこの双曲線対角線の下にローカライズされているので、これは、MPCのを簡単に識別することができます。 1つの異なるMPCのサブコンポーネントは、二次元SDS - PAGEの縦線で区切られています。いくつかのdinstinctのMPCはの構成要素であるコンポーネントは、MPCの大きさに応じて水平線を識別することができます。しかし、それを垂直方向に1行に現れるいくつかのタンパク質のスポットがまたBN - PAGEで同じ位置に移行する独立した複合体の一部であることを考慮する必要があります。彼らは同じMPC内に存在すること、最終的な証拠は抗体ベースのゲルシフトアッセイによって得ることができます。このアッセイでは、細胞溶解物は、前のBN - PAGEに同定されたスポットの一つで表されるタンパク質に対する抗体とともにインキュベートされる。最初の次元の高い分子量の方にこのタンパク質を含むすべてのMPCのシフトでこの結果。また、これらのMPCの一部である他のタンパク質は、この複雑な固有のシフトを受けるため、二次元目SDS -ゲルで簡単に識別できるでしょう。 MPCはの組成はBN - PAGEで分析だけでなく、それらの化学量論の決定が可能です（SchamelとReth 2000）することができるだけではなく、（Schamel 2001）、（スワミー、Minguet ら2007）。この目的のために、NAMOSアッセイ（ネイティブ抗体ベースの移動度シフトアッセイ）を行うことができます。抗体ベースのゲルシフトアッセイと同様に、細胞溶解物は、モノクローナルサブユニット特異的抗体とインキュベートされています。これは、MPCは化学量論にシフトの程度から推論を可能にするBN -ゲルで電気泳動immunoshiftsの誘導につながる conlusionでは、BN - PAGEは、MPCはの同定とそれらのサイズ、組成だけでなく、相対的な豊かさの測定に適しています。 NAMOSアッセイとして実行、それはまた、特定のMPCの化学量論を決定する可能性を提供しています。その一般的な適用を考えると、このテクニックは、MPCはの特性評価（。デッカー、ミュラーら 1996）は非常に有益なツールです、（ウィッティッヒとSchagger 2008）;（ワーグナー、Rehling ら2009。）、（ウィッティッヒとSchägger 2009） 。

blue native polyacrylamide gel electrophoresis (bn-page) for analysis of multiprotein complexes from cellular lysates

ほとんどのタンパク質が他のタンパク質と機能的または規制上の複合体（サリ、Glaeser ら、2003）で見つけることができるので、多タンパク質複合体（MPCは）、細胞シグナル伝達において重要な役割を果たしている。従って、タンパク質 - タンパク質相互作用ネットワークの研究は、タンパク質の機能や制御の統合的な理解を得るためのMPCの詳細な特性評価が必要になります。識別と分析のために、MPCは、ネイティブ条件下で区切る必要があります。このビデオでは、我々は青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）でのMPCの分析について説明します。 BN - PAGE、ゲル濾過やショ糖密度遠心分離によって提供されるよりも高い解像度で本来のコンフォメーションでのMPCの分離を可能にする技術であり、そしてMPCのサイズ、組成、および相対量（SchäggerとフォンJagow 1991）を決定するために有用です。 ;（Schägger、クラマーら1994。）。この方法によって、タンパク質は、ポリアクリルアミドのマトリックスで、その流体力学的サイズと形状に応じて分離されています。ここで、我々は、ライセート透析は、BN - PAGEこれらの生物学的試料に適用するために重要なステップであることを指摘し、全細胞溶解液ののMPCの分析を示しています。最初の次元BN -および二次元目SDS - PAGEの組み合わせを使用して、我々は、BN - PAGEによって分離MPCは、さらにSDS - PAGEによって彼らの個々の成分に分割することができることを示している。ゲルの分離時にMPCのコンポーネントの可視化は、標準的な免疫ブロッティングにより実行されます。 BN - PAGEによるMPCの解析の例として、我々は十分に特徴付け真核生物の19S、20S、および26Sプロテアソームを選んだ。

Watch this Scientific Journal Video about Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates at JoVE.com

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

このビデオでは、我々は青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）により、多タンパク質複合体（MPCは）の特性について説明します。最初の次元では、透析の細胞溶解物を、個々のMPCを識別するために、BN - PAGEによって分離される。二次元目SDS - PAGEにおいては、関心のMPCは、さらに免疫ブロッティングにより、その成分を分析するために細分化されています。

細胞溶解物からの多タンパク質複合体の解析のための青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

blue-native-polyacrylamide-gel-electrophoresis-bn-page-for-analysis

Research

JoVE Journal

Biology

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

130.3K ビュー.  University of Freiburg. このビデオでは、我々は青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）により、多タンパク質複合体（MPCは）の特性について説明します。最初の次元では、透析の細胞溶解物を、個々のMPCを識別するために、BN - PAGEによって分離される。二次元目SDS - PAGEにおいては、関心のMPCは、さらに免疫ブロッティングにより、その成分を分析するため?...

ビデオ: Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

変性剤濃度勾配ゲル電気泳動（DGGE）

生物学

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

セミ変性洗剤-アガロースゲル電気泳動によりアミロイド凝集のためにスクリーニング

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

変性尿素ポリアクリルアミドゲル電気泳動（尿素PAGE）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

DNA断片の分離のためのアガロースゲル電気泳動

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

天然のタンパク質からアミロイドの迅速な生成<em&gt;インビトロ</em

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

に基づいた多タンパク質複合体の比較分析のための新たなアプローチ<sup&gt; 15</sup&gt; N代謝標識および定量的質量分析

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

細胞壁の弾性特性のAFMベースのマッピング：ティッシュ、携帯電話、および細胞内の解像度で

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

ムチンアガロースゲル電気泳動：高分子量の糖タンパク質のためのウェスタンブロッティング


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

タンデムアフィニティータグ法を用いた構造解析のためのネイティブ複合体の精製

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

ブルー ネイティブ ポリアクリルアミドゲル電気泳動法と免疫ブロットを使用して培養されたひと細胞のミトコンドリア呼吸鎖複合体の解析

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...