1. Préparation de protéines de l&#39;échantillon <ol><li> Dans un tube de 500 ul microcentrifugeuse, combiner les éléments suivants: </li></ol><table border="1"><tbody><tr><td> Réactifs </td><td> Volume d&#39;ajouter: </td></tr><tr><td> * Échantillon de protéines (jusqu&#39;à 1 mg) </td><td> Jusqu&#39;à 112 uL </td></tr><tr><td> Tampon d&#39;échantillon (5X) fourni en cartouche kit </td><td> 30 uL </td></tr><tr><td> TNT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> x uL d&#39;un total de volume d&#39;échantillon de 150μL ** </td></tr></tbody></table><ol start="2"><li> Dénaturer les échantillons à 95 ° C pendant 5 minutes. Laisser refroidir à température ambiante. </li></ol> * Le montant total de la protéine d&#39;être séparé dépendra de la complexité de l&#39;échantillon. Pour des échantillons plus complexes, tels que homogénat tissulaire, 150-200 mg par canal est recommandée. ** Si le volume de l&#39;échantillon est inférieure à 112 uL, ajouter 18 Ω H 2 O pour amener le volume final à 150 pi. 2. Chargement de la cartouche GELFREE <ol><li> Retirez la cartouche GELFREE 8100 du sachet en aluminium. </li><li> Retirez et jetez le scellant la plaque. </li><li> Retirez le tampon de stockage de l&#39;compartiments de la cartouche à l&#39;aide d&#39;une pipette. Si les 8 chambres de la cartouche sera utilisée, la cartouche peut être inversée pour drainer le tampon de stockage des compartiments. </li><li> Préparation de la cartouche pour le chargement en ajoutant GELFREE Tampon Courir pour chacune des chambres cartouche comme suit: </li></ol><table border="1"><tbody><tr><td> GELFREE Cartouche Chambre </td><td> Volume de tampon GELFREE Exécution d&#39;ajouter </td></tr><tr><td> Réservoir tampon d&#39;anode </td><td> 8 ml </td></tr><tr><td> Réservoir tampon cathode </td><td> 6 ml </td></tr><tr><td> Collection chambres </td><td> 100 uL de chaque </td></tr></tbody></table><ol start="5"><li> Retirez et jetez les tampons de la chambre de chargement des échantillons. </li><li> Chargez les 150 échantillons uL dans les chambres de chargement à l&#39;aide une pipette à 8 canaux. </li><li> Placez la cartouche chargée dans la Station GELFREE Fractionnement 8100. Bas matrices d&#39;électrodes et fermer le couvercle. </li></ol> 3. Exécution de la cartouche <ol><li> En utilisant l&#39;affichage de l&#39;instrument s à écran tactile, appuyez sur le bouton méthode pour afficher la liste des méthodes préprogrammées. Chacune des méthodes pré-programmées se compose de tensions et de mettre en fonction du temps des pauses visant à résoudre les protéines au sein d&#39;une gamme spécifique de poids moléculaires. Le 8100 GELFREE automatiquement une pause pour la collecte de fractions à des intervalles spécifiés dans la méthode. </li><li> Une fois la méthode appropriée a été identifié, appuyez sur le bouton de récupérer, et entrez le numéro de l&#39;désiré pré-programmés méthode utilisant le clavier à l&#39;écran. Appuyez sur OK, puis appuyez sur le bouton Appliquer. La méthode qui a été appliquée apparaîtra au bas de l&#39;écran principal. </li><li> Pour sélectionner les canaux (nombre d&#39;échantillons) qui seront utilisées dans la course, appuyez sur le bouton de canal sur l&#39;écran principal. Pour sélectionner l&#39;ensemble des huit canaux, appuyez sur sélectionner tous. Ensuite, appuyez sur Terminé pour revenir à l&#39;écran principal. </li><li> Assurez-vous que le couvercle de protection est fermée et que le voyant est vert au bas de l&#39;écran. </li><li> Pour commencer la séparation des protéines, appuyez sur Start. La station de fractionnement sera automatiquement en pause quand il est temps de rassembler les fractions. Note: Bien que le système est en marche, les cercles indiqué dans la boîte sur le côté gauche de l&#39;écran représentent l&#39;état de chaque canal. Un cercle vert indique que le canal est actif, un cercle jaune indique que l&#39;instrument a fait une pause, et un cercle rouge indique que le canal a échoué. Les courants et tensions appliqués sont affichés à droite des cercles état ​​du canal. Les fractions ne doit être retiré lorsque le statut de cercles ont viré au jaune et une alerte de texte apparaît sur ​​l&#39;écran de l&#39;instrument de s. </li><li> Pour enlever les fractions, ouvrez le couvercle de l&#39;instrument et l&#39;utilisation d&#39;une pipette à 8 canaux pour retirer 150 pi de chacun des bassins de collecte. </li><li> Une fois les fractions ont été enlevés, se laver la chambre de collecte deux fois avec du tampon du film GELFREE en ajoutant 100 ul par canal et le pipetage haut et en bas deux fois. </li><li> Après le lavage, ajouter 100 ul d&#39;GELFREE Tampon retour dans les chambres de collecte, de fermer le couvercle et appuyez sur le bouton de reprise. </li><li> Le GELFREE 8100 se déroulera jusqu&#39;au prochain intervalle de temps. Le temps total restant dans l&#39;expérience est montré sur le côté droit de l&#39;écran. Pour basculer entre le temps restant, le temps écoulé et le temps de pause, appuyez sur le bouton ci-dessus la boîte affichant le temps. </li><li> Une fois toutes les fractions ont été collected, ils peuvent être immédiatement utilisés pour la préparation en aval, tels que la focalisation isoélectrique ou électrophorèse sur gel, ou pour l&#39;analyse des liquides en utilisant la spectrométrie de masse chromatographie ou d&#39;immunoaffinité. </li></ol> 4. Les résultats représentatifs Un mg d&#39;homogénat de foie 200 bovins a été divisée en 12 fractions chacun, en utilisant le système de fractionnement GELFREE. 6 uL de chaque fraction a été exécuté sur un gel PAGE 1D SDD et colorés à l&#39;argent. Comme le montre la figure 1, l&#39;échantillon de protéine a été fractionné en 12 fractions distinctes, couvrant toute la gamme de poids moléculaire de 150kDa 3.5kDa d&#39;. Un standard de poids moléculaire (ColorBurst, Sigma) sont également indiquées dans les lignes 1 et 14. Le gel présentés ici met en évidence le taux élevé de recouvrement réalisé en utilisant les GELFREE 8100 du système de fractionnement des protéines. Le gel montré ici représente 4% de la protéine récupéré à partir de 200 ug. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. Les protéines de foie de boeuf homogénat séparés en utilisant le système de fractionnement GELFREE. 6 uL de chaque fraction 150 pi a été chargé et exécuté sur un gel SDS-PAGE 1D et colorés à l&#39;argent. Un poids moléculaire (MW) la norme a également été chargé comme indiqué. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">cliquez ici</a> pour voir une version agrandie de la figure 1.

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

The authors of this article are employed by Protein Discovery that produces reagents and instruments used in this article.

Le système de fractionnement GELFREE 8100 fournit une méthode rapide et simple et reproductible pour le partitionnement des échantillons de protéines complexes en fonction du poids moléculaire discrète fractions. Cette nouvelle méthode de fractionnement élimine le besoin de bande ou de coupe au comptant, ce qui permet d&#39;isoler les protéines sur une gamme large masse en 90 minutes. La possibilité de charger des quantités relativement importantes de l&#39;échantillon et de récupérer les fractions en phase liquide est particulièrement utile dans l&#39;isolement et l&#39;analyse des protéines de faible abondance, qui sont généralement plus difficiles à détecter. Les fractions obtenues peuvent ensuite être utilisés dans un large éventail d&#39;applications, telles que la LC-MS, MALDI-TOF, et immunoblot.

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

Le système de fractionnement GELFREE 8100 est un système de nouvelle protéine fractionnement conçu pour maximiser la récupération des protéines au cours du fractionnement poids moléculaire basée. Le système est composé d&#39;une seule utilisation, les cartouches capacité de 8-échantillon et un instrument de paillasse Fractionnement GELFREE. Durant la séparation, une tension constante est appliquée entre l&#39;anode et la cathode réservoirs, et sur chaque mélange protéine est électrophorétiquement entraînés par une chambre de chargement dans une colonne de gel gel spécialement conçu. Les protéines sont concentrées dans une bande étroite dans un gel de concentration, et séparés en fonction de leurs mobilités électrophorétiques respectives dans un gel de résolution. Comme les protéines éluées de la colonne, ils sont piégés et concentré en phase liquide dans la chambre de collecte, libre du gel. L&#39;instrument est alors interrompue à intervalles de temps spécifiques, et les fractions sont recueillies à l&#39;aide d&#39;une pipette. Ce processus est répété jusqu&#39;à ce que toutes les fractions désirées ont été recueillies. Si moins de 8 échantillons sont analysés sur une cartouche, toute chambres inutilisées peuvent être utilisées dans les séparations ultérieures. Cette nouvelle technologie facilite la séparation simple et rapide de jusqu&#39;à 8 mélanges complexes de protéines simultanément, et offre plusieurs avantages comparativement aux méthodes de fractionnement disponibles auparavant. Ce système est capable de fractionnement jusqu&#39;à 1 mg de protéines totales par canal, pour un total de 8 mg par cartouche. Protéines intactes sur une gamme large masse sont séparés sur la base du poids moléculaire, en conservant importantes propriétés physico-chimiques de l&#39;analyte. Le piégeage en phase liquide prévoit le recouvrement élevé tout en éliminant le besoin de bande ou de coupe endroit, rendant le procédé de fractionnement très reproductible 1.

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

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

La vidéo d&#39;accompagnement décrit l&#39;utilisation du système de fractionnement GELFREE 8100, les partitions des échantillons protéiques complexes sur la base du poids moléculaire et récupère les fractions en phase liquide. La vidéo décrit comment la technologie fonctionne, comment il est utilisé, et fournit des données résultantes, avec analyse par électrophorèse en gel de polyacrylamide d&#39;homogénat de foie fractionnée bovine.

Utiliser le système de fractionnement GELFREE 8100 pour les Poids Moléculaire à base de fractionnement avec récupération de la phase liquide

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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16.3K vues.  Protein Discovery, Inc.. La vidéo d'accompagnement décrit l'utilisation du système de fractionnement GELFREE 8100, les partitions des échantillons protéiques complexes sur la base du poids moléculaire et récupère les fractions en phase liquide. La vidéo décrit comment la technologie fonctionne, comment il est utilisé, et fournit des données résultantes, avec analyse par électrophorèse en gel de polyacrylamide d'homogénat de foie fractionnée bovine.

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

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

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

Préparation et fractionnement de Xenopus laevis oeufs Extraits

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

Recherche

Biologie

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

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

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

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

En fractionnement subcellulaire situ des adhérentes et non-adhérents des cellules de mammifères

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

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

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

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

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

Visualisation des Caenorhabditis elegans</emOuvrages cuticules> Utilisation du colorant lipophile Vital, Dii

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

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

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

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

Do-It-Yourself dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

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

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

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

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

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-Nanobodies

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

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Marquage métabolique et la membrane de fractionnement pour comparative Analyse protéomique de<em&gt; Arabidopsis thaliana</em&gt; Cultures Suspension cellulaires

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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

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

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

Polysome Fractionnement et analyse des mammifères Translatomes sur une échelle de l&#39;ensemble du génome

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

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

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

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

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

L&#39;étude de la protéine système de contrôle de la qualité des<em&gt; D. discoideum</em&gt; Utilisation de cellules vivantes d&#39;imagerie à température contrôlée

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

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

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

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

Fractionnement subcellulaire pour l’Activation de ERK par traitement de peptides dérivés mitochondriale

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

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

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

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

Enclos de graphène de cellules de mammifères chimiquement fixes pour microscopie électronique en phase liquide

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