Este trabalho foi financiado pelo NIH concede R01NS046835 e R01AG034389, ea March of Dimes conceder 6FY08-322. Um agradecimento especial é dedicado a Sra. Eleanor Brown (Miltenyi Biotec, Auburn, CA), que ajudou tremendamente com a sua visão sobre a tecnologia MACS. Miltenyi generosamente forneceu o material utilizado para a demonstração dos experimentos, sem nenhum custo. Agradeço também ao Dr. Guanghu Wang (Medical College of Geórgia / Georgia Health Sciences University, Augusta, GA), que gerou o PKCζ expressar linhas celulares. Apoio por parte do Instituto de Medicina Molecular da Faculdade de Medicina da Geórgia / Georgia Health Sciences University (sob direção do Dr. Lin Mei) também é reconhecido.

1. Introdução A cromatografia de afinidade lipídica mediada por vesículas usando magnético-activated cell sorting (LIMACS) técnica foi desenvolvida em nosso laboratório para isolar complexos ceramida-proteína associada 1-3. Originalmente, as vesículas lipídicas eram feitas de ceramida e fosfatidilserina, o que permitiu a MACS usando partículas magnéticas conjugado com anexina V (altamente afim de fosfatidilserina) para isolar as vesículas e suas proteínas associadas. Temos usado a técnica para o LIMACS na reconstituição in vitro de um complexo de polaridade ceramida-associado eo isolamento de ceramida proteínas de ligação de célula lisados ​​3. LIMACS pode ser modificada usando outros parceiros de interação para o isolamento das vesículas (por exemplo, glicolipídeo-specifc lectinas ou anticorpos lipídica). 2. Procedimentos experimentais Preparação de vesículas lipídicas e Ensaios aPKCBinding <ol><li> Vesículas lipídicas são obtidos a partir de misturas secas de quantidades equimolares de fosfatidilserina (105 mg) e C16-ceramida (85 mcg), seguindo procedimentos modificados para a preparação de lipossomas grandes 1,4-7. </li><li> As misturas de lipídios são, então, ressuspenso e sonicado por 1 h em 100 l de tampão vesícula consistindo de 50 mM Tris / HCl (pH 7,5) e 150 mM NaCl. </li><li> Depois de adicionar 300 ml de tampão vesícula suplementado com 0,1 mM MnCl 2, as amostras são centrifugadas a 12.000 g por 20 min μ a 4 ° C. </li><li> O pellet (vesículas lipídicas grande) é ressuspenso em 100 ul de tampão vesícula e incubadas com 1 nmol de Vybrant CM-dii por 1 hora a 37 ° C para visualizar a fração vesícula após a separação MACS. Vybrant CM-DII é um corante vermelho fluorescente especificamente incorporando em membranas lipídicas. </li><li> Um detergente livres lisado celular é preparado por sonicação / homogeneização das células em 300 ml de tampão hipotônico (10 mM Tris / HCl (pH 7,0) com protease e os inibidores da fosfatase), seguida pela remoção de detritos membranosa por centrifugação. A etapa de centrifugação a 100.000 xg por 1 h deve ser adicionado para evitar a contaminação do lisado celular com membranas endógena contendo fosfatidilserina. </li><li> O lisado limpo é adicionado à suspensão vesícula de lipídios, ea mistura é incubada por 2 horas a 4 ° C. </li><li> A mistura de reação é complementado com 20 l de tampão 20x anexina V vinculativo e 50 ul de uma solução contendo esferas magnéticas conjugadas a anexina V seguido de incubação por 1 hora a 4 ° C. </li><li> MACS é realizada de acordo com as instruções do fabricante (Miltenyi Biotec, Inc.) protocolo. A presença ea quantidade de vesículas lipídicas é determinada através do monitoramento da fluorescência CM-dii Vybrant nas frações de fluxo e eluição utilizando uma leitora de microplacas de fluorescência. </li><li> A correlação linear entre a quantidade de lipídios vesicular e intensidade de fluorescência da vesícula-bound Vybrant CM-dii é verificada pela quantitativa de alto desempenho cromatografia em camada fina (HPTLC) da mistura de lipídios aplicada a anexina V-Macs. </li><li> A especificidade da reação de ligação de proteínas aPKC ou outros, para as vesículas ceramida / fosfatidilserina é verificada por um ensaio de competição de anticorpos usando 1 mg de anticorpo anti-coelho PKCζ policlonais para incubar o lisado celular por 1 hora a 4 ° C antes da incubação com os lipídios vesículas. </li><li> A proteína de ligação a ceramida / fosfatidilserina vesículas no eluato MACS é analisada por SDS-PAGE e immunoblotting. </li></ol> In vitro complexa polaridade lipídios proteínas <ol><li> A reconstituição in vitro de um complexo de proteína-lipídica polaridade é realizada seguindo o procedimento LIMACS como descrito na seção anterior. Em resumo, fosfatidilserina (420 mg) e C16-ceramida (107 mg) é seco a partir de solvente orgânico. </li><li> Os lipídios são secas sob sonicação ressuspenso em 500 mL de tampão vesícula (50 mM Tris / HCl, pH 7,5, 150 mM NaCl). </li><li> Cinco mL de 10 mM MnCl2, 1 ml de Vybrant CM-DII, e 500 ng de PKCζ (recombinante humana) é adicionada ea mistura de reação incubada undergentle agitação por 60 min a 4 ° C. </li><li> Vybrant CM-dii manchada fosfatidilserina / ceramida vesículas são recuperadas por centrifugação a 12.000 xg por 60 min a 4 ° C. </li><li> O pellet (rosa) é ressuspenso em 100 mL tampão Tris e suplementado com γS-GTP (100 mM), o PIB (1 mM), GST-Par6 (100 ng) ou GST-Cdc42 (500 ng) e mais incubadas por 3 h, a 4 ° C (qualquer outra combinação de proteínas recombinantes de interesse pode ser usado aqui). </li><li> Anexina V-tampão (5 mL de uma solução estoque 20x) e anexina V conjugada esferas magnéticas (50 ul) são adicionados a mistura de reação e incubado sob agitação suave por mais 30 min a 4 ° C. </li><li> Anexina V-MACS é realizada seguindo o protocolo do fornecedor, conforme descrito anteriormente. </li&gt; <li> A fração de eluição (1 ml) é suplementado com 10 mg de ovalbumina puro como ajuda de precipitação. A proteína é concentrado por Wessel-Flügge precipitação e analisados ​​por SDS-PAGE/immunoblotting como descrito anteriormente 8. </li><li> A quantidade de vesículas lipídicas eluída é quantificado pela detecção de Vybrant CM-dii (rosa) na fase orgânica (clorofórmio metanol /) da reação de precipitação Wessel Flügge. A quantidade de proteína analisado é normalizado em quantidades iguais de vesículas lipídicas. </li></ol> 3. Resultados LIMACS purificação de PKCζ-EGFP e do domínio de ligação de ceramida C20ζ-EGFP A lisado detergente livres de células MDCK expressar comprimento total PKCζ C-terminal ligada à proteína verde fluorescente (EGFP-FLζ) ou um domínio de ligação de ceramida no C-terminal de PKCζ (C20ζ-EGFP) foi incubada com fosfatidilserina / vesículas ceramida como descrito em procedimentos experimentais. Após eluição da coluna de MACS, proteína foi analisada através immunoblotting e anticorpos contra PKCζ e EGFP para detecção da proteína eluída 2. <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="Figura 1" /> Figura 1. LIMACS de EGFP-rotulados PKCζ e seu fragmento C-terminal C20ζ usando fosfatidilserina / ceramida vesículas. Detergente livres de lisados ​​de células MDCK expressando EGFP (como um controle não vinculativo), comprimento total PKCζ-EGFP, ou a ligação de ceramida, C-terminal de fragmento C20ζ-EGFP foram incubadas com fosfatidilserina / vesículas ceramida tal como descrito na seção de procedimentos experimentais . Depois de usar LIMACS, a proteína foi eluída com tampão de amostra SDS e analisadas por SDS-PAGE e immunoblotting. O painel esquerdo mostra que EGFP não se ligam às vesículas retidos com o V-linked anexina esferas magnéticas. O painel do meio e da direita mostra que o comprimento total PKCζ-EGFP e C20ζ-EGFP foram retidos devido à ligação de ceramida.

<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>

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Para testar a interação específica entre um lipídio e sua proteína de ligação é prejudicada pela incorporação de lipídios na membrana da célula. A membrana celular consiste de uma mistura de lipídios e proteínas diversas e é organizado em microdomínios lipídicos ou jangadas. Portanto, o co-purificação de proteínas microdomínios e não pode distinguir claramente se uma proteína diretamente liga-se a um lipídio ou só é enriquecido em uma estrutura microdomínio. Outros métodos usando lipídios de...

Anexina V conjugada esferas magnéticas e MACS micro e minicolunas foram fornecidos pela Miltenyi Biotec, Inc. (Auburn, CA). Todos os lipídios foram de maior pureza e obtido a partir de Lipídeos Avanti Polar (Alabaster, AL).

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

A análise de interação proteína lipídica é difícil porque os lipídios são incorporados nas membranas celulares e, portanto, inacessíveis para a maioria dos procedimentos de purificação. Como alternativa, os lipídios podem ser revestidos em superfícies planas como as usadas em lipídios ELISA e ressonância Plasmon espectroscopia. No entanto, lipídios revestimento de superfície não formam estruturas microdomínio, que pode ser importante para as propriedades de lipídios vinculativo. Além disso, estes métodos não permitem a purificação de grandes quantidades de proteínas de ligação a lipídeos seu alvo. Para superar essas limitações dos testes de interação de proteínas lipídios e purificar proteínas de ligação de lipídios foi desenvolvido um novo método denominado de lipídios cromatografia de afinidade vesícula mediada magnética usando-activated cell sorting (LIMACS). Neste método, vesículas lipídicas são preparados com os lipídios alvo e fosfatidilserina como os lipídios âncora para anexina V MACS. Fosfatidilserina é um fosfolipídio de membrana onipresente celular que mostra alta afinidade à proteína anexina V. Usando esferas magnéticas conjugadas a anexina V vesículas lipídicas contendo fosfatidilserina-se ligará à esferas magnéticas. Quando as vesículas lipídicas são incubadas com uma célula lisado a proteína de ligação ao lipídios alvo também será obrigado a as contas e pode ser co-purificado utilizando MACS. Este método também pode ser usado para testar se proteínas recombinantes reconstituir um complexo de proteínas de ligação ao lipídios alvo. Temos utilizado este método para mostrar a interação da PKC atípica (aPKC) com a ceramida sphingolipid e co-purificar resposta a apoptose de próstata 4 (PAR-4), uma proteína de ligação a ceramida associada aPKC. Temos também utilizado este método para a reconstituição de um complexo de ceramidas associados da aPKC recombinante com a célula de polaridade proteínas relacionadas Par6 e Cdc42. Desde vesículas lipídicas pode ser preparado com uma variedade de esfingo ou fosfolipídios, LIMACS oferece um teste versátil para lipídios proteínas de interação em um ambiente de lipídios que se assemelha de perto o da membrana celular. Complexos de proteína adicional de lipídios pode ser identificado através da análise proteômica de proteínas lipídios vinculativo co-purificada com as vesículas lipídicas.

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

Para testar a interação de uma proteína com o seu alvo de lipídios foi utilizado MACS e anexina V conjugada esferas magnéticas e vesículas de lipídios sintetizados a partir de lipídios alvo e fosfatidilserina anexina V vinculativo. Proteínas vinculado à lipídica alvo são co-purificados e analisados ​​após eluição dos grânulos.

Cromatografia de afinidade lipídica mediada por vesículas com separação de células Magnetic Ativado (LIMACS): um novo método para analisar proteínas lipídios Interação

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

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Biology

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

12.4K visualizações.  Georgia Health Sciences University. Para testar a interação de uma proteína com o seu alvo de lipídios foi utilizado MACS e anexina V conjugada esferas magnéticas e vesículas de lipídios sintetizados a partir de lipídios alvo e fosfatidilserina anexina V vinculativo. Proteínas vinculado à lipídica alvo são co-purificados e analisados ​​após eluição dos grânulos.

Vídeo: 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.

Separação de células de fluorescência Activated de Protoplastos Usina

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

Biologia

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.

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

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.

Avidez baseado Triagem Interação Extracelular (AVEXIS) para a detecção escalável de baixa afinidade Extracelular receptor-ligante Interações

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.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

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.

Análise de célula específico<em&gt; Arabidopsis</em&gt; Folhas utilizando ordenação células activadas por fluorescência

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.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

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.

Combinando Sorting magnética de células mãe e testes de flutuação para Analisar Genoma Instabilidade Durante Mitotic envelhecimento celular em<em&gt; Saccharomyces cerevisiae</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.

Purificação de Native Complexos de estudo estrutural usando um método Tag Tandem Affinity

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.

Magnético-ativado da pilha classificação estratégias para isolar e purificar sinovial fluidos derivados mesenquimais células-tronco de um modelo de coelho

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.

Novo método de entrega de DNA do plasmídeo para Mouse bexiga Urotélio por eletroporação

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