1。サンプルのタンパク質の調製<ol><li> 500μlの微量遠心チューブに、次のように組み合わせる。 </li></ol><table border="1"><tbody><tr><td>試薬</td><td>追加するボリューム： </td></tr><tr><td> *タンパク質のサンプル（最大1mg）を</td><td>最大112μLまで</td></tr><tr><td> カートリッジキットに付属のサンプルバッファー（5X） </td><td> 30μL </td></tr><tr><td> DTT 1M </td><td> 8μL </td></tr><tr><td> 18ΩH 2 O </td><td> 150μLのサンプル量の合計**からxμL </td></tr></tbody></table><ol start="2"><li> ℃で5分間、95℃の試料の変性。室温まで冷却。 </li></ol> *分離されるタンパク質の総量は、サンプルの複雑さに依存します。このような組織ホモジネートなどのより複雑なサンプルについては、チャンネルあたり150から200μgのをお勧めします 。 **サンプル量がより小さい112μLの場合は、150μLに最終的なボリュームをもたらすために、18Ω、H 2 Oを加える。 2。 GELFREEカートリッジのロード<ol><li>箔の袋からGELFREE 8100カートリッジを取り外します。 </li><li>プレートシーラーを取り外して廃棄。 </li><li>ピペットを使用してカートリッジの各コンパートメントからストレージのバッファを削除します。カートリッジの全8室が使用される場合、カートリッジは区画からストレージのバッファをドレインに反転させることができます。 </li><li>次のようにカートリッジ室の各々にGELFREEランニングバッファーを追加してロードするためのカートリッジを準備します。 </li></ol><table border="1"><tbody><tr><td> GELFREEカートリッジ商工会議所</td><td>追加するGELFREEランニングバッファーの量</td></tr><tr><td>陽極バッファータンク</td><td> 8 mLの</td></tr><tr><td>陰極バッファータンク</td><td> 6 mLの</td></tr><tr><td>コレクション室</td><td> 100μL各</td></tr></tbody></table><ol start="5"><li>サンプルのローディング室から任意のバッファを削除し、捨てる。 </li><li> 8チャンネルピペッターを使用してロードのチャンバーに150μLのサンプルをロードします。 </li><li> GELFREE 8100分別ステーションにロードされたカートリッジを置きます。電極アレイを下げて、蓋を閉じます。 </li></ol> 3。カートリッジの実行<ol><li>楽器のタッチスクリーンディスプレイを使用して、事前にプログラムされたメソッドのリストを表示する方法のボタンを押してください。事前にプログラムの各メソッドは、分子量の特定の範囲内のタンパク質を解決するために設計された一連の電圧と時間ベースのポーズで構成されています。 GELFREE 8100はメソッドで指定された間隔でフラクションコレクションのために自動的に一時停止します。 </li><li>一旦、適切なメソッドが識別されて、 取得ボタンを押すと、画面上のキーパッドを用いて所望のプリプログラム方法の番号を入力します。 [OK]を押して、[ 適用]ボタンを押してください。適用されたメソッドは、メイン画面の下部に表示されます。 </li><li>実行時 ​​に使用されるチャネル（サンプル数）を選択するには、メイン画面上のチャンネルボタンを押してください。すべての8つのチャネルを選択するには、 すべてを選択キーを押します。次に、メイン画面に戻りますDoneを押します。 </li><li>安全蓋が閉じていることを確認し、インジケータライトが画面の下部にある緑色であること。 </li><li>タンパク質の分離を開始するには、 スタートを押してください 。それは画分を集めた時期になると分別ステーションは自動的に一時停止される注：システムが動作している間、画面の左側にあるボックスに示されている円は、各チャネルの状態を表しています。緑色の円は、チャネルがアクティブであることを示し、黄色の円は、機器が一時停止していることを示し、赤い円は、チャネルが失敗したことを示します。印加される電圧と電流はチャネルステータス円の右側に表示されます。ステータス円が黄色になっているとテキストのアラートが機器の画面上に表示されたときの画分をのみ削除する必要があります。 </li><li>分画を削除するには、楽器の蓋を開き、コレクションのチャンバーのそれぞれから150μLを撤回する8チャンネルピペットを使用してください。 </li><li>分画が削除されたら、チャンネルあたり100μLを追加し、二回、上下にピペッティングすることによりGELFREEランニング緩衝液で2回収集チャンバーを洗浄する。 </li><li>洗浄後、コレクション室に戻ってバッファを実行GELFREEの100uL追加、蓋を閉め、 再開ボタンを押してください。 </li><li> GELFREE 8100は次の時間間隔になるまで実行されます。実験に残っている合計時間が画面の右側に表示されます。 残り時間を切り替えるには、時間が経過し、一時停止の時間、時間を表示するボックスの上のボタンを押してください。 </li><li>一度画分のすべてを同じ場所にされているllectedは、彼らはすぐにこのような等電点電気泳動やゲル電気泳動などのさらなる下流の準備、、または液体クロマトグラフィー質量分析法または免疫親和性を用いて分析するために使用することができます。 </li></ol> 4。代表的な結果ウシ肝臓ホモジネートの200μgのはGELFREE分別システムを使用して、12分画ごとに分割されました。各画分の6μLを標準1D SDS PAGEゲルと銀染色上で実行された。図1に示すように、タンパク質サンプルは3.5kDaから150kDaに分子量の範囲にまたがる、12種類の画分に分画した。分子量の標準は、（カラーバースト、シグマ）またレーン1及び14に示されています。ここに示されているゲルは、タンパク質の分画にGELFREE 8100システムを使用して達成高回収率を強調。ここに示されているゲルは、200μgのから回収したタンパク質の4％を表します。 <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="図1" border="0" /> 図1。 GELFREE分画システムを使用して分離されたウシ肝臓のホモジネートからのタンパク質が各 150μL分画の6μLは、1D SDS PAGEゲルと銀染色でロードされ、実行されました。示されるように分子量（MW）標準にもロードされました。してください<a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。

<ol>
	<li>Tran, J. C., Doucette, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Size+Separation+of+Intact+Proteins+in+Solution+Phase+for+Mass+Spectrometry.">Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry.</a> Anal Chem.. , (2009).</li></ol>

この記事の著者は、この記事で使用した試薬と器具を生産するタンパク質の探索によって採用されています。

GELFREE 8100分別システムは、離散的な分子量ベースのフラクションに複雑なタンパク質サンプルを分割するための迅速、簡単かつ再現性のある方法を提供する。分別のこの新しい方法により、90分で広い質量範囲にわたってタンパク質を分離すること、バンドやスポットをカットする必要がなくなります。サンプルの比較的大量をロードし、液相での分画を回収する能力は、通常、検出が難しい低濃度タンパク質の単離と解析に特に有用です。得られた画分は、その後、LC - MS、MALDI - TOF、およびイムノブロッティングなどの幅広い用途で使用できます。

using the gelfree 8100 fractionation system for molecular weight-based fractionation with liquid phase recovery

GELFREE 8100分別システムは、分子量に基づく分画中のタンパク質の回収を最大化するよう設計された新規タンパク質分画システムです。システムは、シングルユース、8サンプルの容量のカートリッジとベンチトップGELFREE分画音源で構成されています。分離時には、一定の電圧が陽極と陰極リザーバとの間に印加されると、各タ​​ンパク質の混合物を電気泳動的に特別に設計されたゲルカラムゲルにロードするチャンバーから駆動されます。タンパク質がスタッキングゲルでタイトなバンドに集中し、そして分離ゲルのそれぞれの電気泳動移動度に基づいて分離されている。カラムから溶出するタンパク質として、彼らが閉じ込められたと収集チャンバ内の液相に集中し、ゲルの自由 。楽器は、特定の時間間隔で一時停止され、そして画分をピペットを用いて収集されます。すべての所望の画分が回収されるまで、このプロセスが繰り返されます。未満の8サンプルがカートリッジ上で実行されている場合、未使用の任意のチャンバーはその後の分離に使用することができます。 この新技術は、同時に最大8複雑なタンパク質混合物の迅速かつ簡単な分離を容易にし、以前に利用可能な分画法と比較した場合、いくつかの利点を提供しています。このシステムは、カートリッジあたり8mgの合計で、チャネルあたりの総タンパク質1mgの、最大の分画が可能です。広い質量範囲にわたって無傷のタンパク質は、分析対象物の重要な物理化学的性質を保持し、分子量に基づいて分離されています。バンドまたはスポットカットの必要性を排除しながら、液相の巻き込みは、1分画のプロセスは、再現性の高いこと、高い回復のために用意されています。

Watch this Scientific Journal Video about Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery at JoVE.com

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

付属のビデオでは、分子量に基づいてGELFREE 8100分画システムの使用、パーティションの複雑なタンパク質サンプルを説明し、液相での分画を回収する。ビデオは、それが使用されているか、技術がどのように動作するかについて説明し、分画されたウシ肝臓のホモジネートのポリアクリルアミドゲル電気泳動分析で、結果のデータを提供します。

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 ...

using-gelfree-8100-fractionation-system-for-molecular-weight-based

Research

JoVE Journal

Biology

16.3K ビュー.  Protein Discovery, Inc.. 付属のビデオでは、分子量に基づいてGELFREE 8100分画システムの使用、パーティションの複雑なタンパク質サンプルを説明し、液相での分画を回収する。ビデオは、それが使用されているか、技術がどのように動作するかについて説明し、分画されたウシ肝臓のホモジネートのポリアクリルアミドゲル電気泳動分析で、結果のデータを提供します。

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

アフリカツメガエル卵抽出物の調製と分画

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

生物学

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

の可視化線虫（Caenorhabditis elegans）</em親油性生体染色色素、DIIを使用する>キューティクルの構造

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

ゲノム規模で哺乳類Translatomesのソーム分画および分析

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

タンパク質の品質管理システムを学びます<em&gt; D。 discoideumの</em&gt;温度制御された生細胞イメージングを用い

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

ミトコンドリア由来ペプチド治療時に ERK の活性化のための細胞レベル下の分別

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

液相電子顕微鏡用化学固定哺乳類細胞のグラフェンエンクロージャ

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...