Este trabajo fue apoyado por el NIH y R01NS046835 R01AG034389, y la March of Dimes conceder 6FY08-322. Un agradecimiento especial se dedica a la Sra. Eleanor Brown (Miltenyi Biotec, Auburn, CA), que ayudó enormemente con su visión de la tecnología MACS. Miltenyi ha ofrecido generosamente el material utilizado para la demostración de los experimentos, sin costo alguno. También estoy agradecida con el Dr. Wang Guanghu (Medical College of Georgia / Georgia Universidad Ciencias de la Salud, Augusta, GA), que generó la PKCζ líneas celulares que expresan. Apoyo del Instituto de Medicina Molecular de la Facultad de Medicina de Georgia / Ciencias de la Salud de la Universidad de Georgia (bajo dirección del Dr. Lin Mei) también se reconoce.

1. Introducción Los lípidos de vesículas mediada por cromatografía de afinidad utilizando magnético activado por la selección de células (LIMACS) técnica fue desarrollada en nuestro laboratorio para aislar la ceramida asociada a complejos de proteínas 1.3. Originalmente, las vesículas lipídicas se hicieron de la ceramida y la fosfatidilserina, lo que permitió el uso de partículas magnéticas MACS-anexina V conjugada (muy afín a la fosfatidilserina) para aislar las vesículas y sus proteínas asociadas. Hemos utilizado la técnica de LIMACS para la reconstitución in vitro de un complejo de ceramidas polaridad asociada y el aislamiento de la ceramida proteínas de unión a partir de lisados ​​celulares 3. LIMACS se puede modificar mediante otros socios de interacción para el aislamiento de las vesículas (por ejemplo, glicolípidos-specifc lectinas y anticuerpos de lípidos). 2. Los procedimientos experimentales Preparación de vesículas de lípidos y Ensayos aPKCBinding <ol><li> Vesículas de lípidos se obtienen a partir de mezclas secas de cantidades equimolares de fosfatidilserina (105 mg) y C16-ceramida (85 mg) después de procedimientos modificados para la preparación de liposomas grandes 1,4-7. </li><li> Las mezclas de lípidos después se volvieron a suspender y se sonica durante 1 h en 100 l de tampón de vesículas que consta de 50 mM Tris / HCl (pH 7,5) y 150 mM NaCl. </li><li> Después de la adición de 300 l de tampón vesícula suplementado con 0,1 mM de MnCl2, las muestras se centrifugan a 12.000 g μ por 20 min a 4 ° C. </li><li> El pellet (vesículas grandes lípidos) se resuspende en 100 l de buffer de la vesícula y se incubaron con 1 nmol de Vybrant CM-dii durante 1 hora a 37 ° C para visualizar la fracción de vesículas después de la separación MACS. Vybrant CM-dii es un tinte rojo fluorescente específicamente incorporar en las membranas lipídicas. </li><li> Un detergente libre de lisado celular se prepara por ultrasonidos / homogeneización de las células en 300 l de tampón hipotónico (10 mM Tris / HCl (pH 7.0) con la proteasa y los inhibidores de la fosfatasa) seguido de la eliminación de los desechos membranosa por centrifugación. Un paso de centrifugación a 100.000 xg durante 1 h, debe añadirse a evitar la contaminación del lisado celular con membranas endógeno que contiene fosfatidilserina. </li><li> El lisado aclarado se añade a la suspensión de vesículas de lípidos, y la mezcla se incuba durante 2 horas a 4 ° C. </li><li> La mezcla de reacción se complementa con 20 l de buffer 20x anexina V vinculante y 50 l de una solución que contiene partículas magnéticas conjugadas con anexina V seguido de la incubación durante 1 hora a 4 ° C. </li><li> MACS se realiza de acuerdo a las instrucciones del fabricante (Miltenyi Biotec, Inc.) de protocolo. La presencia y la cantidad de vesículas de lípidos está determinada por el control de la Vybrant CM-dii de fluorescencia en las fracciones a través del flujo y elución utilizando un lector de microplacas de fluorescencia. </li><li> La correlación lineal entre la cantidad de lípidos vesicular y la intensidad de fluorescencia de la vesícula con destino Vybrant CM-dii es verificada por cuantitativos de alto rendimiento cromatografía en capa fina (HPTLC) de la mezcla de lípidos se aplica a la anexina V-Macs. </li><li> La especificidad de la reacción de unión de proteínas aPKC u otras a las vesículas ceramida / fosfatidilserina es verificada por un ensayo de competición de anticuerpos por medio de 1 mg de anti-PKCζ anticuerpo policlonal de conejo para la incubación del lisado celular durante 1 hora a 4 ° C antes de la incubación con los lípidos vesículas. </li><li> La proteína de unión a vesículas ceramida / fosfatidilserina en el eluido MACS se analizaron por SDS-PAGE e inmunoblotting. </li></ol> En el complejo de la polaridad de los lípidos in vitro de proteínas <ol><li> La reconstitución in vitro de un complejo lípido-proteína de la polaridad se realiza siguiendo el procedimiento LIMACS como se describe en la sección anterior. En breve fosfatidilserina, (420 mg) y C16-ceramida (107 mg), se seca a partir de un disolvente orgánico. </li><li> Los lípidos secos se volvieron a suspender en tratamiento con ultrasonidos en 500 l de buffer de vesícula (50 mM Tris / HCl, pH 7,5, 150 mM NaCl). </li><li> Cinco l de 10 mM MnCl2, 1 l de Vybrant CM-DII, y 500 ng de PKCζ (recombinante humana) se añade la mezcla de reacción se incubó undergentle agitación durante 60 min a 4 ° C. </li><li> Vybrant CM-dii manchada fosfatidilserina / ceramida vesículas se recuperó por centrifugación a 12.000 xg durante 60 min a 4 ° C. </li><li> El pellet (rosa) se resuspende en 100 l de tampón Tris y se complementa con γS-GTP (100 M), el PIB (1 mM), GST-PAR6 (100 ng), o GST-Cdc42 (500 ng) y se incubaron durante 3 horas a 4 ° C (cualquier otra combinación de proteínas recombinantes de interés puede ser utilizado aquí). </li><li> Anexina V-buffer (5 l de una solución madre 20 veces) y anexina V conjugada con partículas magnéticas (50 l) se añadió la mezcla de reacción se incubaron en agitación suave durante 30 min a 4 ° C. </li><li> Anexina V-MACS se realiza siguiendo el protocolo del proveedor como se describió anteriormente. </li&gt; <li> La fracción de elución (1 ml) se complementa con 10 mg de albúmina de huevo pura como la ayuda de precipitación. La proteína se concentra por Wessel-Flugge precipitación y analizados por SDS-PAGE/immunoblotting como se describió previamente 8. </li><li> La cantidad de vesículas de lípidos eluidos se cuantifica mediante la detección de Vybrant CM-DII (rosa) en los orgánicos (cloroformo / metanol) fase de la reacción de precipitación Wessel Flugge. La cantidad de proteína analizada se normaliza en la misma cantidad de vesículas de lípidos. </li></ol> 3. Resultados LIMACS purificación de PKCζ-EGFP y el dominio de unión de la ceramida C20ζ-EGFP Un lisado con detergente libre de células MDCK expresar toda su longitud PKCζ C-terminal ligada a la proteína verde fluorescente (EGFP FLζ-) o un dominio de unión de la ceramida en el C-terminal de PKCζ (C20ζ-EGFP) se incubó con fosfatidilserina / vesículas ceramida como se describe en los procedimientos experimentales. Tras la elución de la columna de MACS, la proteína se analizó mediante inmunotransferencia y anticuerpos contra PKCζ y EGFP para la detección de la proteína eluye 2. <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="Figura 1" /> Figura 1. LIMACS de EGFP-etiquetados PKCζ y su C-terminal del fragmento C20ζ utilizando fosfatidilserina / ceramida vesículas. Detergente libre de lisados ​​de células MDCK que expresan EGFP (como control no vinculante), la longitud total PKCζ-EGFP, o la unión de la ceramida, C-terminal del fragmento C20ζ-EGFP se incubaron con fosfatidilserina / vesículas ceramida como se describe en la sección de Procedimientos experimentales . Después de usar LIMACS, la proteína se eluyó con tampón de muestra SDS y se analizaron por SDS-PAGE e inmunoblotting. El panel izquierdo muestra que EGFP no se unen a las vesículas mantenido con la anexina V-vinculados partículas magnéticas. El panel del medio y la derecha muestra que la longitud total PKCζ-EGFP y C20ζ-EGFP fueron retenidos debido a la unión a la 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>

Para probar la interacción específica entre un lípido y su unión a proteínas se ve obstaculizada por la incrustación de los lípidos de la membrana celular. La membrana celular consiste en una mezcla de lípidos y proteínas de varios y se organiza en microdominios lipídicos o balsas. Por lo tanto, la co-purificación de microdominios y las proteínas no se pueden distinguir claramente si una proteína se une directamente a un lípido o sólo se enriquece en una estructura de microdominios. Otros métodos que uti...

Anexina V conjugada con perlas magnéticas y micro MACS y minicolumnas fueron proporcionados por Miltenyi Biotec, Inc. (Auburn, CA). Todos los lípidos de alta pureza y la obtenida de Avanti Polar Lipids (Alabaster, AL).

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

El análisis de la interacción lípido proteína es difícil debido a los lípidos están incrustados en las membranas celulares y, por tanto, inaccesible para la mayoría de los procedimientos de purificación. Como alternativa, los lípidos pueden estar recubiertas por superficies planas que se utilizan para lípidos ELISA y resonancia de plasmones de la espectroscopia. Sin embargo, los lípidos de la superficie de revestimiento no forman microdominios estructuras, que pueden ser importantes para las propiedades de unión de lípidos. Además, estos métodos no permiten la purificación de grandes cantidades de proteínas de unión a los lípidos de su objetivo. Para superar estas limitaciones de las pruebas de interacción de proteínas y lípidos para purificar las proteínas de unión de lípidos, hemos desarrollado un nuevo método denominado vesícula lipídica mediada por cromatografía de afinidad utilizando magnético activado por la selección de células (LIMACS). En este método, las vesículas de lípidos son preparados con el objetivo de lípidos y la fosfatidilserina como los lípidos de anclaje para anexina V MACS. Fosfatidilserina es un fosfolípido de la membrana celular omnipresente que muestra una alta afinidad a la proteína anexina V. Con perlas magnéticas conjugadas con anexina V de las vesículas de lípidos que contienen fosfatidilserina se unen a las bolas magnéticas. Cuando las vesículas lipídicas se incuban con un lisado celular de la proteína de unión a los lípidos objetivo también tendrá la obligación de las cuentas y puede ser co-purificado utilizando MACS. Este método también se puede utilizar para comprobar si las proteínas recombinantes reconstruir un complejo de proteínas de unión a los lípidos de destino. Hemos utilizado este método para mostrar la interacción de la PKC atípicas (aPKC) con la ceramida esfingolípidos y co-purificar respuesta apoptosis de próstata 4 (PAR-4), una proteína de unión a la ceramida asociada aPKC. También hemos utilizado este método para la reconstitución de un complejo de ceramidas asociados de aPKC recombinante con la célula relacionados con la polaridad de las proteínas PAR6 y Cdc42. Desde las vesículas de lípidos pueden ser preparados con una variedad de sphingo o fosfolípidos, LIMACS ofrece una prueba versátil para la interacción lípido-proteína en un ambiente de lípidos que se parece mucho a la de la membrana celular. Otros complejos de lípidos y proteínas pueden ser identificadas mediante análisis de proteómica de proteínas de unión de lípidos co-purifica con las 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 probar la interacción de una proteína con su objetivo de lípidos se utilizó MACS y anexina V conjugada con partículas magnéticas y de las vesículas de lípidos sintetizados a partir de los lípidos de destino y fosfatidilserina anexina V vinculante. Proteínas unidas a los lípidos de destino se co-purificados y analizados después de la elución de las cuentas.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

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 vistas.  Georgia Health Sciences University. Para probar la interacción de una proteína con su objetivo de lípidos se utilizó MACS y anexina V conjugada con partículas magnéticas y de las vesículas de lípidos sintetizados a partir de los lípidos de destino y fosfatidilserina anexina V vinculante. Proteínas unidas a los lípidos de destino se co-purificados y analizados después de la elución de las cuentas.

Video: 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.

Fluorescencia de células activadas por la clasificación de los protoplastos de la planta

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

Investigación

Biología

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.

El aislamiento de lábil Multi-complejos de proteínas por In vivo Control Celular entrecruzamiento y Inmuno-cromatografía de afinidad magnética

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 detección basada en la interacción extracelular (AVEXIS) para la detección escalable de extracelular de baja afinidad interacciones ligando-receptor

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.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-Nanobodies

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.

Análisis específico de célula de<em&gt; Arabidopsis</em&gt; Hojas usando citometría de flujo

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 manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

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 clasificación magnética de las células madre y las pruebas de fluctuación para analizar la inestabilidad del genoma Durante mitótico Aging Cell en<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.

Purificación de Complejos nativo para el estudio estructural se utiliza un 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.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

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.

Novedoso método de plásmido ADN entrega al urotelio de la vejiga de ratón por electroporación

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