この作品は、NIHの助成金R01NS046835とR01AG034389、そしてダイム助成6FY08 - 322の月によってサポートされていました。特別あなたがMACS技術に彼女の洞察力で大いに役立った夫人エレノアブラウン（Miltenyi Biotec社、オーバーン、カリフォルニア州）に捧げられる感謝。ミルテニーは、気前よく無料での実験のデモンストレーションのために使用される材料を提供しています。私はまた、細胞株の発現PKCζを生成博士広滬王（グルジア/グルジア健康科学大学の医科大学、オーガスタ、ジョージア州）に感謝しています。グルジア/グルジア健康科学大学（リン博士は梅の指揮下）の医科大学分子医学研究所のサポートも認められている。

1。はじめ磁気活性化セルソーティング（LIMACS）技法を用いて脂質小胞を媒介としたアフィニティークロマトグラフィーは、セラミド結合蛋白質複合体1-3を分離するために我々の研究室で開発されました。もともと、脂質の小胞は小胞とその関連蛋白質を分離するために磁性粒子標識アネキシンVを（ホスファチジルセリンに非常にアフィン）を使用してMACSに許可されるセラミド、ホスファチジルセリン、から成っていた。セラミド関連極性複合体と細胞のセラミド結合タンパク質の分離の in vitro再構成に 3を溶解液のために我々はLIMACS技術を使用している。 LIMACSは、小胞の分離（例えば、糖脂質- specifcレクチンまたは脂質抗体）のために他の相互作用パートナーを使用して変更することができます。 2。実験手順脂質小胞とaPKCBindingアッセイの準備<ol><li>脂質小胞は、ホスファチジルセリン（105μg）を、大規模なリポソーム調製1,4-7のために修飾された手順に従ってC16 -セラミド（85μg）の等モル量の乾燥混合物から得られる。 </li><li>脂質の混合物はその後、50mMトリス/ HCl（pH7.5）及び150mMのNaClで構成される小胞のバッファー100μlの中で1時間再懸濁し、超音波処理されています。 </li><li> 0.1mMのMnCl 2を補充した小胞のバッファー300μlを添加した後、試料を4時20分、12,000μg を ℃で遠心分離されています</li><li>ペレットは、（大脂質小胞）° C MACS分離した後、小胞の割合を視覚化する小胞のバッファー100μlに再懸濁し、37℃で1時間Vybrant CM - DII、1 nmolのでインキュベートする。 Vybrant CM - DIIは、特に脂質膜に組み込んだ赤色蛍光色素です。 </li><li>界面活性剤を含まない細胞溶解液を遠心分離によって膜破片の除去に続いて低張緩衝液（プロテアーゼ及びホスファターゼ阻害剤との10mMトリス/ HCl（pH7.0）で）を300μlの超音波/細胞の均質化によって調製される。 1時間、100,000 × gで遠心分離工程は、ホスファチジルセリンを含む内在性の膜と細胞溶解液の汚染を避けるために追加する必要があります。 </li><li>清澄化ライセートは、脂質のベシクル懸濁液に添加され、そして混合物を4℃で2時間インキュベートする</li><li>反応混合物を20倍のアネキシンV結合バッファーとアネキシンVに結合した磁気ビーズを含む溶液50μlを20μlと補足されては、4℃で1時間インキュベートした</li><li> MACSは、製造業者の（ミルテニーバイオテク社）プロトコルに従って行われます。脂質小胞の存在と量は、マイクロプレート蛍光リーダーを用いてフロースルーと溶出分画のVybrant CM - DIIの蛍光をモニターすることによって決定されます。 </li><li>水疱性脂質の量と小胞に結合Vybrant CM - DIIの蛍光強度との間の線形相関は、アネキシンV -ベースのMacに適用される脂質の混合物の定量的な高性能薄層クロマトグラフィー（HPTLC）によって検証されます。 </li><li>セラミド/ホスファチジルセリン小胞にaPKCまたは他のタンパク質の結合反応の特異性は、℃のインキュベーションの前に4℃で1時間細胞ライセートをインキュベートする抗PKCζウサギポリクローナル抗体1μgを用いて抗体の競合アッセイによって検証され脂質小胞。 </li><li> MACS溶出液中のセラミド/ホスファチジルセリン小胞への結合タンパク質は、SDS - PAGEとイムノブロッティングによって分析されます。 </li></ol> 体外脂質-タンパク質極性複雑で <ol><li>脂質-タンパク質の極性複合体の in vitro再構成の前のセクションで説明したようにLIMACSの手順に従って実行されます。簡単に言えば、ホスファチジルセリン（420μg）をとC16 -セラミドは、（107μg）を有機溶剤から乾燥させる。 </li><li>乾燥脂質は小胞の緩衝液500μl（; 150mMのNaCl 50mMトリス/ HCl、pH7.5）に超音波処理下で再懸濁する。 </li><li> 10 mMのMnCl2、Vybrant CM - DIIを1μl、およびPKCζの500ngの（ヒト組み換え）の5μlを加え、反応混合物を4℃で60分間undergentleの攪拌をインキュベートする℃に</li><li> Vybrant CM - DIIステンドホスファチジルセリン/セラミド小胞は4℃で60分間℃、12,000 xgで遠心分離によって回収されています</li><li>ペレットは、（ピンク）100μlのTris緩衝液に再懸濁し、γS- GTP（100μM）を補充し、GDP（1 mM）を、GST - Par6（100 ng）を、またはGST - Cdc42と（500 ngの）と、さらに3インキュベートする4にあるh ° C（ 目的の組換えタンパク質の他の組み合わせをここで使用することができます ）。 </li><li>アネキシンV -バッファー（20倍ストック溶液を5μl）とアネキシンVが結合した磁気ビーズ（50μl）を加え、反応混合物を4℃でさらに30分間℃で穏やかに撹拌しながらインキュベートする。 </li><li>アネキシンV - MACSは、前述のように供給者のプロトコルに従って行われます。 </li&gt; <li>溶出画分（1ml）を沈殿助剤としての純粋な卵白アルブミン10μgを補足されています。タンパク質は、ヴェッセル-フリューゲェ沈殿により濃縮し、以前に8を説明するようにSDS-PAGE/immunoblottingによって分析されます。 </li><li>溶出された脂質小胞の量は、ヴェッセルフリューゲェの沈殿反応の有機（クロロホルム/メタノール）相におけるVybrant CM - DII（ピンク）の検出によって定量化される。分析されたタンパク質の量は、脂質小胞の同量で正規化されます。 </li></ol> 3。結果 LIMACSのPKCζ- EGFPの精製とセラミド結合ドメインC20ζ- EGFP 界面活性剤を含まないPKCζC -末端に緑色蛍光タンパク質（FLζ- EGFP）またはPKCζ（C20ζ- EGFP）のC末端におけるセラミド結合ドメインにリンクされている全長を発現しているMDCK細胞のライセートは、ホスファチジルセリン/セラミド胞などと一緒にインキュベートした実験手順で説明しています。 MACSカラムの溶出後、タンパク質は、イムノブロットおよび溶出したタンパク質2の検出のためのPKCζとEGFPに対する抗体を用いて解析した。 <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="図1" /> の図1。LIMACS EGFP標識PKCζとそのC -末端断片C20ζホスファチジルセリン/セラミド小胞を用いた。 実験手順の項で説明するようにEGFP（非結合制御など）、全長PKCζ- EGFP、またはセラミド結合、C -末端断片C20ζ- EGFPを発現するMDCK細胞の界面活性剤を含まない溶解物は、ホスファチジルセリン/セラミドの小胞とインキュベートした。 。 LIMACSを使用した後、タンパク質をSDSサンプルバッファーで溶出され、SDS - PAGEおよび免疫ブロッティングにより分析した。左側のパネルでは、EGFPは、アネキシンV -リンクされた磁気ビーズを保持小胞に結合していないことを示しています。真ん中と右のパネルはその完全な長さを示すPKCζ- EGFPとC20ζ- EGFPは、セラミドへの結合のために保持された。

<ol>
	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

利害の衝突は宣言されません。

脂質とその結合タンパク質間の特異的な相互作用をテストすることは細胞膜の脂質から埋め込むことによって妨げられている。細胞膜には、いくつかの脂質とタンパク質の混合物から成り、それは、脂質ミクロドメインまたは筏で構成されています。蛋白質が直接脂質に結合するかだけミクロドメイン構造に富んでいるそのため、マイクロドメインとタンパク質の共精製は、はっきりと区別?...

アネキシンV -共役磁気ビーズとMACSマイクロとminicolumnsはミルテニーバイオテク株式会社（オーバーン、カリフォルニア州）から提供された。すべての脂質は、最高純度のものであったとAvanti極性脂質（アラバスター、AL）から入手。

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

脂質はほとんどの精製操作にアクセスできない、したがって、細胞膜に埋め込まれているため、脂質タンパク質の相互作用の解析は困難です。別の方法として、脂質は脂質ELISAとプラズモン共鳴分光法のために使われる平らな表面上に塗布することができる。しかし、表面コーティングの脂質は、脂質結合特性のために重要であるかもしれない、ミクロドメイン構造を形成しない。さらに、これらのメソッドは、それらの標的脂質に結合する蛋白質の大量の精製のために許可されていません。 脂質のタンパク質相互作用をテストするのこれらの限界を克服し、脂質結合タンパク質を精製するために、我々は磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティークロマトグラフィーと呼ばれる新しい方法を開発した。この方法では、脂質小胞は、アネキシンV MACSのためのアンカーの脂質のようなターゲットの脂質、ホスファチジルセリンを用いて調製されています。ホスファチジルセリンはタンパク質アネキシンに高い親和性を示すVのホスファチジルセリン含有脂質の小胞は、磁性ビーズに結合するアネキシンVに結合した磁気ビーズを用いたユビキタス細胞膜のリン脂質である。脂質の小胞がライセート、細胞とインキュベートされている場合、ターゲット脂質への結合タンパク質はまた、ビーズに結合され、MACSを用いて共同で精製することができる。この方法は、組換えタンパク質の再構成する場合、対象脂質に結合するタンパク質複合体をテストするために使用することができます。 我々は、スフィンゴ脂質セラミドとし、共同浄化前立腺アポトーシス応答4（PAR - 4）、セラミド関連aPKCの結合タンパク質への非定型PKC（aPKC）との相互作用を表示するには、このメソッドを使用している。我々はまた、細胞極性関連タンパク質Par6およびCdc42との組換えaPKCのセラミド関連複合体の再構築のためにこのメソッドを使用している。脂質小胞がsphingo -またはリン脂質のさまざまな準備ができるので、LIMACSはその細胞膜の酷似脂質環境における脂質 - タンパク質相互作用のための多目的なテストを提供しています。追加の脂質蛋白質複合体は、脂質結合タンパク質、脂質小胞との共精製のプロテオミクス解析を用いて同定することができる。

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

そのターゲットの脂質とタンパク質の相互作用をテストするために我々は、MACSとターゲットの脂質とアネキシンV結合ホスファチジルセリンから合成されたアネキシンV -共役磁気ビーズと脂質小胞を使用。ターゲットの脂質に結合したタンパク質は、共同で精製し、ビーズから溶出した後に分析されます。

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

lipid-vesicle-mediated-affinity-chromatography-using-magnetic

Research

JoVE Journal

Biology

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

12.4K ビュー.  Georgia Health Sciences University. そのターゲットの脂質とタンパク質の相互作用をテストするために我々は、MACSとターゲットの脂質とアネキシンV結合ホスファチジルセリンから合成されたアネキシンV -共役磁気ビーズと脂質小胞を使用。ターゲットの脂質に結合したタンパク質は、共同で精製し、ビーズから溶出した後に分析されます。

ビデオ: Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物プロトプラストの蛍光活性化細胞選別

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

生物学

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

不安定な多タンパク質複合体の単離で in vivoで制御セルラー架橋及び免疫磁気アフィニティークロマトグラフィー

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

の細胞特異的な分析<em&gt;シロイヌナズナ</em&gt;蛍光活性化細胞選別を使用した葉

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

母細胞と変動試験の磁気ソーティングを組み合わせることでの有糸分裂細胞老化中に、ゲノム不安定性を分析するために<em&gt;サッカロマイセス·セレビシエ</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

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 ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

磁気活性化細胞を分離し、滑膜液由来間葉系幹細胞が家兎モデルからの浄化戦略を並べ替え

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...