我々は、彼らのアドバイスやコメントのための彼のサポートとGeula Davidov、ノームGrimbergと陳グットマンのために博士アミールAharoniを認めます。

<ol>
	<li>Faivre, D., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+Bacteria+and+Magnetosomes.">Magnetotactic Bacteria and Magnetosomes.</a> Chem Rev. 108, 4875-4898 (2008).</li><li>D'Andrea, L. D., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TPR+proteins:+the+versatile+helix.">TPR proteins: the versatile helix.</a> Trends Biochem Sci. 28, 655-662 (2003).</li><li>Young, J. C., Barral, J. M., Hartl, U. l. r. i. c. h., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+folding:+localized+functions+of+cytosolic+chaperones.">More than folding: localized functions of cytosolic chaperones.</a> Trends Biochem Sci. 28, 541-547 (2003).</li><li>Brocard, C., Hartig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+targeting+signal+1:+is+it+really+a+simple+tripeptide?.">Peroxisome targeting signal 1: is it really a simple tripeptide?.</a> Biochim Biophys Acta. 1763, 1565-1573 (2006).</li><li>Fransen, M., Amery, L., Hartig, A., Brees, C., Rabijns, A., Mannaerts, G. P., Van Veldhoven, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+PTS1-+and+Rab8b-binding+properties+of+Pex5p+and+Pex5Rp/TRIP8b.">Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b.</a> Biochim Biophys Acta. 1783, 864-873 (2008).</li><li>Baker, M. J., Frazier, A. E., Gulbis, J. M., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein-import+machinery:+correlating+structure+with+function.">Mitochondrial protein-import machinery: correlating structure with function.</a> Trends Cell Biol. 17, 456-464 (2007).</li><li>Mirus, O., Bionda, T., von Haeseler, A., Schleiff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionarily+evolved+discriminators+in+the+3-TPR+domain+of+the+Toc64+family+involved+in+protein+translocation+at+the+outer+membrane+of+chloroplasts+and+mitochondria.">Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.</a> J Mol Model. 15, 971-982 (2009).</li><li>Gatsos, X., Perry, A. J., Anwari, K., Dolezal, P., Wolynec, P. P., Likic, V. A., Purcell, A. W., Buchanan, S. K., Lithgow, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+and+outer+membrane+assembly+in+Alphaproteobacteria.">Protein secretion and outer membrane assembly in Alphaproteobacteria.</a> FEMS Microbiol Rev. 32, 995-1009 (2008).</li><li>Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B., Nandicoori, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+residues+in+Mycobacterium+tuberculosis+protein+kinase+G+play+a+role+in+regulating+kinase+activity+and+survival+in+the+host.">Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.</a> J Biol Chem. 284, 27467-27479 (2009).</li><li>Edqvist, P. J., Broms, J. E., Betts, H. J., Forsberg, A., Pallen, M. J., Francis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetratricopeptide+repeats+in+the+type+III+secretion+chaperone,+LcrH:+their+role+in+substrate+binding+and+secretion.">Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.</a> Mol Microbiol. 59, 31-44 (2006).</li><li>Grunberg, K., Muller, E. C., Otto, A., Reszka, R., Linder, D., Kube, M., Reinhardt, R., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+proteomic+analysis+of+the+magnetosome+membrane+in+Magnetospirillum+gryphiswaldense.">Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense.</a> Appl Environ Microbiol. 70, 1040-1050 (2004).</li><li>Komeili, A., Vali, H., Beveridge, T. J., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proc Natl Acad Sci USA. 101, 3839-3843 (2004).</li><li>Okuda, Y., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+a+magnetosome-associated+protein,+TPR-containing+MAM22,+in+Escherichia+coli.">Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.</a> FEBS Lett. 491, 169-173 (2001).</li><li>Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. F., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localizations+of+Mam22+and+Mam12+in+the+magnetosomes+of+Magnetospirillum+magnetotacticum.">Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.</a> J Bacteriol. 188, 3805-3812 (2006).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li></ol>

タンパク質の精製は、任意のタンパク質生化学的または構造的な研究の主要なステップです。それぞれの蛋白質がそれ自身の動作と一意であるため、人はそのプロパティを定義し、それに応じてその精製を変更する必要があります。蛋白質のターゲットは、バイオインフォマティクスのツールを使用して精製するための第一歩として分析する必要があります。彼らは、環境と特殊なイオン/配...

<table><tbody><tr><td>French Press</td><td>Equipment</td><td>Thermo Fisher Scientific, Inc.</td><td>FA-078A</td><td></td></tr><tr><td>Pressure cell</td><td>Equipment</td><td>Thermo Fisher Scientific, Inc.</td><td>FA-032</td><td></td></tr><tr><td>Ultra-centrifuge</td><td>Equipment</td><td>Sorvall, Thermo Scientific</td><td>Discovery 90SE</td><td></td></tr><tr><td>Rottor</td><td>Equipment</td><td>Beckman Coulter Inc.</td><td>Ti60</td><td></td></tr><tr><td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td><td>Equipment</td><td>Beckman Coulter Inc.</td><td>BC-355618</td><td></td></tr><tr><td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td><td>Equipment</td><td>Bio-Rad</td><td>737-2521</td><td></td></tr><tr><td>Ni-NTA His Bind resin</td><td>Equipment</td><td>Novagen, EMD Millipore</td><td>M0063428</td><td></td></tr><tr><td>Spectrophotometer</td><td>Equipment</td><td>Amersham</td><td>Ultraspec 2100 pro</td><td></td></tr><tr><td>Quartz cuvette</td><td>Equipment</td><td>Hellma</td><td>104-QS</td><td></td></tr><tr><td>Fast Performance Liquid Chromatography- AKTA purifier 10</td><td>Equipment</td><td>GE Healthcare</td><td>28-4062-64</td><td></td></tr><tr><td>Ion exchange column &amp;#8211; MonoQ 4.6/100 PE</td><td>Equipment</td><td>GE Healthcare</td><td>10025543</td><td></td></tr><tr><td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td><td>Equipment</td><td>GE Healthcare</td><td>17-1071-01</td><td></td></tr><tr><td>Centricon - Vivaspin15 &amp;#8211; 10,000 MWCO</td><td>Equipment</td><td>Sartorius AG</td><td>VS1501</td><td></td></tr><tr><td>Table centrifuge</td><td>Equipment</td><td>Thermo Fisher Scientific, Inc.</td><td>IEC CL30R</td><td></td></tr><tr><td>MALDI-TOF</td><td>Equipment</td><td>Bruker Corporation</td><td>Reflex IV</td><td></td></tr><tr><td>Tris-HCl (hydrotymethyl) aminomethane</td><td>Reagent</td><td>BioLab</td><td>20092391</td><td></td></tr><tr><td>Sodium Chloride</td><td>Reagent</td><td>FRUTROM</td><td>235553470</td><td></td></tr><tr><td>Imidazole</td><td>Reagent</td><td>Alfa Aesar</td><td>288-32-4</td><td></td></tr><tr><td>EDTA free protease inhibitors cocktail</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P-8849</td><td></td></tr><tr><td>Dnase I (Deoxyribonuclease I)</td><td>Reagent</td><td>Sigma-Aldrich</td><td>DN-25</td><td></td></tr><tr><td>Bovine Thrombin</td><td>Reagent</td><td>Fisher Scientific</td><td>BP25432</td><td></td></tr><tr><td>Glycine</td><td>Reagent</td><td>BioLab</td><td>07132391</td><td></td></tr><tr><td>Soudim Dodecyl Sulfate (SDS)</td><td>Reagent</td><td>BioLab</td><td>19822391</td><td></td></tr><tr><td>Beta-mercapt&amp;#8211;thanol</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M-3148</td><td></td></tr><tr><td>InstantBlue</td><td>Reagent</td><td>Expedeon</td><td>1SB01L</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder</td><td>Reagent</td><td>Fermentas</td><td>SM0671</td><td></td></tr></tbody></table>

purification of the m. magneticum strain amb-1 magnetosome associated protein mama&delta;41

走磁性細菌は、地磁気のフィールドに沿って自分自身をオリエンテーションすることができる水生微生物の多様なグループを構成。この動作は、適切な環境のための彼らの検索を支援するために考えられている<em&gt;（1）</em&gt;。この機能はmagnetosomeにより付与され、脂質の直鎖アセンブリから構成される細胞内小器官は、マグネタイトまたはグレイジャイトの〜50 nmの結晶をbiomineralizeと囲むようにそれぞれができるように小胞。機能的な小胞の形成に必要なことが示されmagnetosomeの主成分はママです。ママは最も特徴付けmagnetosome関連タンパク質の一つである非常に豊富magnetosome関連タンパク質である<em&gt; in vivoで</em&gt;<em&gt;（2-6）</em&gt;。この記事では、ママの浄化に焦点を当てている研究されているにもかかわらず<em&gt; in vivoで</em&gt;、明確な機能的または構造的細部​​は、それのために同定されていない。バイオインフォマティクス解析には、ママがタンパク質を含むテトラ- tricopeptideリピート（TPR）であることが示唆された。 TPRは、蛋白質の広い範囲で大きな倍のようなまたは一部を形成するように見られる構造モチーフである、それはタンパク質 - タンパク質相互作用を媒介多タンパク質複合体のテンプレートとして機能<em&gt;（7）</em&gt;。 TPRsは、真核細胞オルガネラのプロセスと多くの細菌の経路で、多くの重要なタスクに関与している<em&gt;（8-14）。</emママ、タンパク質を含む独自のTPRを理解する&gt;ためには、高度に精製されたタンパク質は、最初のステップとして必要です。この記事では、我々はより安定したママ欠失変異体（MamAΔ41）の精製のプロトコールを提示<em&gt; M. magneticum</em&gt; AMB - 1。

Watch this Scientific Journal Video about Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÎ”41 at JoVE.com

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

ママはmagnetosome活性化に関与することが示されていたユニークなMagnetosome関連するタンパク質である。ここでは、からママ欠失変異体（MamAΔ41）の精製のプロトコールを提示<em&gt; M. magneticum</em&gt; AMB - 1。

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

purification-m-magneticum-strain-amb-1-magnetosome-associated-protein

Research

JoVE Journal

Biology

13.5K ビュー.  Ben-Gurion University. ママはmagnetosome活性化に関与することが示されていたユニークなMagnetosome関連するタンパク質である。ここでは、からママ欠失変異体（MamAΔ41）の精製のプロトコールを提示 M. magneticum AMB - 1。

ビデオ: Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

神経科学

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

完全精製成分を用いたMsp1抽出活性の再構成

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

生化学

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

細胞の超高速磁化のためのカチオン化Magnetoferritinの合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

生物工学

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

アフィニティークロマトグラフィーを用いたヒトマトリックスメタロプロテイナーゼ-3の細菌発現と精製

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

チューニング可能なナノスケールの次元と組成を有するマンガンフェライトクラスターの安定した水性懸濁液

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

化学

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

外因性の抗原クロス プレゼンテーション内の小胞体関連分解膜コンパートメントの精製

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

免疫・感染学

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

当然のことながら環境から磁性細菌の発生の収集、分離、濃縮

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

生物学

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

属磁性の走磁性細菌の成長: MSR 1、AMB 1 MS 1 株

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

食品および環境試料からのサルモネラの準メタゲノム解析

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

2ステップミトコンドリア単離によるタンパク質定量の改善

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

著者スポットライト:タンパク質の発見と定量を改善するためのミトコンドリアの2ステップタグフリー単離(英語)  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

要約

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この動画の章

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

完全精製成分を用いたMsp1抽出活性の再構成

細胞の超高速磁化のためのカチオン化Magnetoferritinの合成

アフィニティークロマトグラフィーを用いたヒトマトリックスメタロプロテイナーゼ-3の細菌発現と精製

チューニング可能なナノスケールの次元と組成を有するマンガンフェライトクラスターの安定した水性懸濁液

外因性の抗原クロスプレゼンテーション内の小胞体関連分解膜コンパートメントの精製

当然のことながら環境から磁性細菌の発生の収集、分離、濃縮

属磁性の走磁性細菌の成長: MSR 1、AMB 1 MS 1 株

食品および環境試料からのサルモネラの準メタゲノム解析

タンパク質の発見と定量を改善するためのミトコンドリアの2段階タグフリー分離

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

要約

さらに動画を探す

この動画の章

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

完全精製成分を用いたMsp1抽出活性の再構成

細胞の超高速磁化のためのカチオン化Magnetoferritinの合成

アフィニティークロマトグラフィーを用いたヒトマトリックスメタロプロテイナーゼ-3の細菌発現と精製

チューニング可能なナノスケールの次元と組成を有するマンガンフェライトクラスターの安定した水性懸濁液

外因性の抗原クロス プレゼンテーション内の小胞体関連分解膜コンパートメントの精製

当然のことながら環境から磁性細菌の発生の収集、分離、濃縮

属磁性の走磁性細菌の成長: MSR 1、AMB 1 MS 1 株

食品および環境試料からのサルモネラの準メタゲノム解析

タンパク質の発見と定量を改善するためのミトコンドリアの2段階タグフリー分離

外因性の抗原クロスプレゼンテーション内の小胞体関連分解膜コンパートメントの精製