1. Preparación de muestras de proteínas <ol><li> En un tubo de microcentrífuga 500 l, combine las siguientes: </li></ol><table border="1"><tbody><tr><td> Reactivo </td><td> Volumen a añadir: </td></tr><tr><td> * La proteína de la muestra (hasta 1 mg) </td><td> Hasta 112 l </td></tr><tr><td> Tampón de muestra (5 veces), siempre en el cartucho de juego </td><td> 30 l </td></tr><tr><td> DTT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> l x a un total de volumen de muestra de 150μL ** </td></tr></tbody></table><ol start="2"><li> Desnaturalizar las muestras a 95 ° C durante 5 minutos. Enfriar a temperatura ambiente. </li></ol> * La cantidad total de proteínas que ha separado dependerá de la complejidad de la muestra. Para muestras más complejas, tales como tejido homogeneizado, 150-200 mg por canal se recomienda. ** Si el volumen de la muestra es inferior a 112 l, agregar 18 Ω H 2 O para llevar el volumen final de 150 mL. 2. Carga del cartucho GELFREE <ol><li> Retire el cartucho de GELFREE 8100 de la bolsa de aluminio. </li><li> Retire y deseche el papel sellador del plato. </li><li> Quitar el tampón de almacenamiento de los compartimentos del cartucho con una pipeta. Si los 8 cámaras del cartucho se utiliza, el cartucho puede ser invertida para drenar el buffer de almacenamiento de los compartimentos. </li><li> Prepare el cartucho para la carga mediante la adición de buffer GELFREE ejecución de cada una de las cámaras de cartucho de la siguiente manera: </li></ol><table border="1"><tbody><tr><td> GELFREE Cartucho Cámara </td><td> Volumen de Buffer GELFREE Running añadir </td></tr><tr><td> Depósito tampón anódico </td><td> 8 ml </td></tr><tr><td> Cátodo tanque de regulación </td><td> 6 mL </td></tr><tr><td> Colección de cámaras </td><td> 100 L cada uno </td></tr></tbody></table><ol start="5"><li> Retire y deseche cualquier buffer de la cámara de carga de la muestra. </li><li> Cargar las 150 muestras l en las cámaras de carga mediante una pipeta de 8 canales. </li><li> Coloque el cartucho cargado en la estación de Fraccionamiento GELFREE 8100. Baje la matrices de electrodos y cierre la tapa. </li></ol> 3. La ejecución del cartucho <ol><li> Usando la pantalla del instrumento s de la pantalla táctil, pulse el botón método para ver la lista de métodos preprogramados. Cada uno de los métodos pre-programados consiste en establecer las tensiones y las pausas en función del tiempo destinado a resolver las proteínas dentro de un rango específico de pesos moleculares. El 8100 GELFREE automáticamente una pausa para la recogida de fracción en los intervalos especificados en el método. </li><li> Una vez que el método apropiado ha sido identificado, presione el botón de recuperar, e introduzca el número deseado de la pre-programados método mediante el teclado en pantalla. Pulse Aceptar y luego pulse el botón Aplicar. El método que se ha aplicado en la parte inferior de la pantalla principal. </li><li> Para seleccionar los canales (número de muestras) que se utilizarán en la ejecución, presione el botón del canal en la pantalla principal. Para seleccionar los ocho canales, pulse Seleccionar todo. A continuación, pulse Listo para volver a la pantalla principal. </li><li> Asegúrese de que la tapa está cerrada y la seguridad de que el indicador es de color verde en la parte inferior de la pantalla. </li><li> Para comenzar la separación de proteínas, pulse Iniciar. La estación de fraccionamiento se detendrá automáticamente cuando sea el momento de recoger las fracciones. Nota: Aunque el sistema está funcionando, los círculos se muestra en el cuadro en la parte izquierda de la pantalla muestran el estado de cada canal. Un círculo verde indica que el canal está activo, un círculo amarillo indica que el instrumento se ha detenido, y un círculo rojo indica que el canal ha fallado. Las tensiones aplicadas y las corrientes se muestran a la derecha de los círculos de estado del canal. Fracciones sólo deben ser retirados cuando el estado de los círculos se han vuelto de color amarillo y una alerta de texto en la pantalla del instrumento s. </li><li> Para eliminar las fracciones, abra la tapa del instrumento y el uso de una pipeta de 8 canales para retirar 150 l de cada una de las cámaras de colección. </li><li> Una vez que las fracciones se han retirado, lavar la cámara de recolección dos veces con tampón GELFREE Correr por la adición de 100 l por canal y pipeteando arriba y abajo dos veces. </li><li> Después del lavado, añadir 100uL de GELFREE funcionamiento de amortiguación de nuevo en las cámaras de recolección, cierre la tapa y pulse el botón Reanudar. </li><li> El GELFREE 8100 se extenderá hasta el siguiente intervalo de tiempo. El tiempo total que queda en el experimento se muestra en la parte derecha de la pantalla. Para alternar entre el tiempo restante, el tiempo transcurrido y el tiempo para hacer una pausa, pulse el botón encima de la caja que muestra la hora. </li><li> Una vez que todas las fracciones se han collected, inmediatamente se puede utilizar para la preparación de más abajo, tales como el isoelectroenfoque y electroforesis en gel, o para el análisis mediante espectrometría de masas cromatografía líquida o de inmunoafinidad. </li></ol> 4. Resultados representante A 200 mg de homogenado de hígado de bovino fue dividida en 12 fracciones cada uno, utilizando el sistema de fraccionamiento GELFREE. 6 l de cada fracción se ejecutan en un gel PAGE 1D SDS y tinción de plata. Como se muestra en la Figura 1, la muestra de proteína se fraccionó en 12 fracciones distintas, que abarcan el rango de peso molecular de 3.5kDa a 150kDa. Un estándar de peso molecular (ColorBurst, Sigma) se muestran también en las calles 1 y 14. El gel se muestra aquí destaca el elevado índice de recuperación de lograr mediante el sistema de GELFREE 8100 para el fraccionamiento de proteínas. El gel se muestra aquí representa el 4% de la proteína recuperada a partir de 200 mg. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Proteínas a partir de hígado de bovino homogenado separado utilizando el sistema de fraccionamiento GELFREE. 6 l de cada fracción de 150 l se carga y se ejecuta en un gel PAGE 1D SDS y tinción de plata. Un peso molecular (MW) estándar se ha cargado también, como se indica. Por favor, <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 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.

El sistema de fraccionamiento GELFREE 8100 proporciona un método rápido, simple y reproducible para la partición de muestras complejas de proteínas en función del peso molecular fracciones discretas. Este nuevo método de fraccionamiento elimina la necesidad de la banda o el corte in situ, por lo que es posible aislar las proteínas en un rango amplio de masas en 90 minutos. La capacidad de carga de cantidades relativamente grandes de la muestra y se recuperan las fracciones en la fase líquida es particularmente útil en el aislamiento y el análisis de proteínas de baja abundancia, que suelen ser más difíciles de detectar. Las fracciones resultantes se pueden utilizar en una amplia gama de aplicaciones, tales como LC-MS, MALDI-TOF, y la inmunotransferencia.

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

El sistema de fraccionamiento GELFREE 8100 es un sistema de fraccionamiento de proteínas novedoso diseñado para maximizar la recuperación de proteínas durante el fraccionamiento en peso, basado molecular. El sistema se compone de un solo uso, de 8 muestras de cartuchos de capacidad y un Instrumento de sobremesa GELFREE fraccionamiento. Durante la separación, una tensión constante se aplica entre el ánodo y el cátodo de embalses, y cada mezcla de proteínas por electroforesis es impulsado a partir de una cámara de carga en un gel en columna de gel especialmente diseñado. Las proteínas se concentran en una banda estrecha en un gel de apilamiento, y separados en función de su movilidad electroforética respectivos en un gel de resolución. Como las proteínas se eluyen de la columna, se encuentran atrapados y se concentra en fase líquida en la cámara de recolección, sin el gel. El instrumento se detuvo en intervalos de tiempo específicos, y las fracciones se recogen con una pipeta. Este proceso se repite hasta que todas las fracciones deseadas se han recogido. Si menos de 8 muestras se realizan en un cartucho, sin usar ninguna cámara se puede utilizar en la separación posterior. Esta nueva tecnología facilita la separación rápida y sencilla de hasta 8 mezclas complejas de proteínas simultáneamente, y ofrece varias ventajas en comparación con los métodos de fraccionamiento previamente disponibles. Este sistema es capaz de fraccionar hasta 1 mg de proteína total por canal, para un total de 8 mg por cartucho. Las proteínas intactas sobre un rango amplio de masas se separan en función del peso molecular, conservando importantes propiedades fisicoquímicas de la sustancia analizada. El atrapamiento fase líquida permite la recuperación de alta al tiempo que elimina la necesidad de una banda de corte o lugar, haciendo que el proceso de fraccionamiento altamente reproducible 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

El video que acompaña describe el uso del sistema de fraccionamiento GELFREE 8100, las particiones que las muestras de proteínas complejas sobre la base del peso molecular y se recupera la fracción en fase líquida. El video describe cómo funciona la tecnología, cómo se utiliza, y proporciona los datos resultantes, con el análisis de electroforesis en gel de poliacrilamida de homogeneizado fraccionada hígado de bovino.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

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

using-gelfree-8100-fractionation-system-for-molecular-weight-based

Research

JoVE Journal

Biology

16.3K vistas.  Protein Discovery, Inc.. El video que acompaña describe el uso del sistema de fraccionamiento GELFREE 8100, las particiones que las muestras de proteínas complejas sobre la base del peso molecular y se recupera la fracción en fase líquida. El video describe cómo funciona la tecnología, cómo se utiliza, y proporciona los datos resultantes, con el análisis de electroforesis en gel de poliacrilamida de homogeneizado fraccionada hígado de bovino.

Video: 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.

Preparación y fraccionamiento de los extractos de huevo Xenopus laevis

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

Investigación

Biología

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 Fraccionamiento subcelular situ de células mamíferas adherentes y no adherentes-

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.

La visualización de Caenorhabditis Elegans Estructuras de la cutícula uso del colorante lipofílico 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 de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

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.

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 ...

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.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

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.

Polisoma Fraccionamiento y análisis de los mamíferos Translatomes en un genoma de gran escala

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.

Estudiar el Sistema de Control de Calidad de la Proteína<em&gt; D. discoideum</em&gt; Uso de imágenes de células vivas con temperatura controlada

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.

Fraccionamiento subcelular para la activación de ERK a péptidos derivados de mitocondrial tratamiento

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.

Carcasa de grafeno de células de mamíferos fijas químicamente para microscopía electrónica de fase líquida

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