1. Preparação de proteínas da amostra <ol><li> Em um tubo de microcentrífuga 500 mL, combine o seguinte: </li></ol><table border="1"><tbody><tr><td> Reagente </td><td> Volume de acrescentar: </td></tr><tr><td> * Amostra de proteína (até 1 mg) </td><td> Até 112 mL </td></tr><tr><td> Tampão de amostra (5X) fornecido em cartucho kit </td><td> 30 mL </td></tr><tr><td> TDT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> x mL para um total de volume de amostra de 150μL ** </td></tr></tbody></table><ol start="2"><li> Desnaturar as amostras a 95 ° C por 5 minutos. Arrefecer à temperatura ambiente. </li></ol> * A quantidade total de proteína a ser separados dependerá da complexidade da amostra. Para amostras mais complexas, tais como tecido homogeneizado, 150-200 mg por canal é recomendado. ** Se o volume da amostra é inferior a 112 mL, adicionar 18 Ω H 2 O para trazer o volume final de 150 mL. 2. Carregar o cartucho GELFREE <ol><li> Remova o cartucho de 8100 GELFREE da embalagem de alumínio. </li><li> Remova e descarte o selador de placa. </li><li> Remover o buffer de armazenamento dos compartimentos do cartucho com uma pipeta. Se todos os 8 câmaras do cartucho será utilizado, o cartucho pode ser invertida para drenar o buffer de armazenamento dos compartimentos. </li><li> Prepare o cartucho para carregar adicionando tampão de corrida GELFREE para cada uma das câmaras do cartucho da seguinte forma: </li></ol><table border="1"><tbody><tr><td> GELFREE Cartucho Câmara </td><td> Volume de GELFREE tampão de corrida para adicionar </td></tr><tr><td> Reservatório de tampão ânodo </td><td> 8 mL </td></tr><tr><td> Catodo reservatório tampão </td><td> 6 mL </td></tr><tr><td> Coleta de câmaras </td><td> 100 L cada </td></tr></tbody></table><ol start="5"><li> Remover e descartar qualquer tampão da câmara de carga da amostra. </li><li> Carregar os 150 amostras mL para as câmaras de carregamento com uma pipeta de 8 canais. </li><li> Coloque o cartucho carregado na Estação Fracionamento GELFREE 8100. Inferiores as matrizes de eletrodos e feche a tampa. </li></ol> 3. Executando o cartucho <ol><li> Utilizar o instrumento s tela sensível ao toque, pressione o botão método para ver a lista de pré-programadas métodos. Cada um dos métodos pré-programados consiste em definir e tensões baseadas em tempo pausas destinadas a resolver as proteínas dentro de uma faixa específica de pesos moleculares. O 8100 GELFREE automaticamente pausa para coleta de fração nos intervalos especificados no método. </li><li> Uma vez que o método adequado foi identificado, pressione o botão recuperar, e digite o número desejado do método de pré-programados usando o teclado na tela. Pressione ok, em seguida, pressione o botão aplicar. O método que tem sido aplicado irá aparecer na parte inferior da tela principal. </li><li> Para selecionar os canais (número de amostras) que será usado na corrida, pressione o botão de canal na tela principal. Para selecionar todos os oito canais, pressione selecionar todos. Em seguida, pressione feito para voltar à tela principal. </li><li> Garantir que a tampa de segurança é fechada e que o indicador está verde na parte inferior da tela. </li><li> Para começar a separação de proteínas, pressione o começo. A Estação de Fracionamento automaticamente pausa quando é hora de coletar frações. Nota: Enquanto o sistema estiver em execução, os círculos mostrados na caixa do lado esquerdo da tela retratam o estado de cada canal. Um círculo verde indica que o canal está ativo, um círculo amarelo indica que o instrumento tem uma pausa e um círculo vermelho indica que o canal tenha falhado. As tensões e correntes aplicadas são exibidos à direita dos círculos status do canal. Frações só deve ser removido quando o status de círculos ficaram amarelas e um alerta de texto aparece na tela do instrumento s. </li><li> Para remover frações, abra a tampa do instrumento e usar uma pipeta de 8 canais para retirar 150 mL de cada uma das câmaras de coleta. </li><li> Uma vez que as frações foram retiradas, lave a câmara de coleta duas vezes com tampão de corrida GELFREE adicionando 100 ul por canal e pipetando para cima e para baixo duas vezes. </li><li> Após a lavagem, adicione 100uL de GELFREE tampão de corrida de volta para as câmaras de coleta, feche a tampa e pressione o botão Continuar. </li><li> O GELFREE 8100 vai decorrer até ao próximo intervalo. O tempo total restante no experimento é mostrado no lado direito da tela. Para alternar entre o tempo restante, tempo decorrido e tempo de pausa, pressione o botão acima da caixa de exibição de tempo. </li><li> Depois de todas as frações foram collected, eles podem ser imediatamente utilizada para a preparação mais a jusante, como a focalização isoelétrica ou eletroforese em gel, ou para análise por espectrometria de massa de cromatografia líquida ou de imunoafinidade. </li></ol> 4. Resultados representante A mcg 200 de homogeneizado de fígado bovino foi dividido em 12 frações de cada um, utilizando o Sistema de Fracionamento GELFREE. 6 mL de cada fração foi executado em um padrão de gel PAGE 1D SDS e corados de prata. Conforme mostrado na Figura 1, a amostra de proteína foi fracionado em 12 frações distintas, abrangendo a faixa de peso molecular de 150kDa para 3.5kDa. Um padrão de peso molecular (Colorburst, Sigma) também são mostrados em faixas 1 e 14. O gel mostrado aqui destaca a taxa de recuperação alta alcançada usando o sistema de 8100 GELFREE para fracionamento de proteínas. O gel mostrado aqui representa 4% da proteína recuperada de 200 mg. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Proteínas de fígado bovino homogeneizado separados usando o sistema de fracionamento GELFREE. 6 mL de cada fração 150 mL foi carregado e executado em um gel 1D PAGE SDS e corados de prata. Um padrão de peso molecular (MW) também foi carregado, conforme indicado. Por favor, <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">clique aqui</a> para ver uma versão ampliada da 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>

Os autores deste artigo são empregados por Descoberta proteína que produz os reagentes e instrumentos utilizados neste artigo.

O Sistema de Fracionamento GELFREE 8100 fornece um método rápido, simples e reprodutível para o particionamento de amostras de proteínas complexas em peso molecular baseados em frações discretas. Esse novo método de fracionamento elimina a necessidade de banda ou corte no local, tornando-se possível isolar proteínas em uma faixa ampla massa em 90 minutos. A capacidade de carregar quantidades relativamente grandes de amostra e recuperar frações em fase líquida é particularmente útil no isolamento e análise de proteínas de baixa abundância, que normalmente são mais difíceis de detectar. As frações resultantes podem então ser usados ​​em uma ampla gama de aplicações, tais como LC-MS, MALDI-TOF e immunoblotting.

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

O Sistema de Fracionamento GELFREE 8100 é um sistema de fracionamento de proteínas romance projetado para maximizar a recuperação de proteínas durante o fracionamento do peso molecular baseado. O sistema é composto de uso único, os cartuchos de oito amostras de capacidade e uma bancada Instrumento Fracionamento GELFREE. Durante a separação, uma tensão constante é aplicada entre o anodo eo catodo reservatórios, e cada mistura de proteínas é eletroforeticamente expulsos de uma câmara de carga em um gel especialmente concebido gel coluna. Proteínas estão concentrados em uma faixa apertada em um gel de empilhamento, e separados com base em suas respectivas mobilidades eletroforéticas em gel de resolução. Como proteínas saem da coluna, eles estão presos e concentrado em fase líquida na câmara de coleta, livre do gel. O instrumento é então fez uma pausa em intervalos de tempo específicos, e as frações são coletados por meio de uma pipeta. Este processo é repetido até que todas as frações desejadas tenham sido coletados. Se menos de 8 amostras são executados em um cartucho, qualquer câmaras não utilizado pode ser utilizado em separações subseqüentes. Esta nova tecnologia facilita a separação rápida e simples de até 8 misturas complexas de proteínas simultaneamente, e oferece diversas vantagens quando comparado aos métodos de fracionamento disponíveis anteriormente. Este sistema é capaz de fracionamento de até 1 mg de proteína total por canal, para um total de 8mg por cartucho. Proteínas intactas em uma faixa de grande massa são separadas com base no peso molecular, mantendo importantes propriedades físico-químicas do analito. O aprisionamento de fase líquida prevê recuperação alta, enquanto eliminando a necessidade de banda ou corte no local, tornando o processo de fraccionamento altamente reprodutível 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

O vídeo que acompanha descreve o uso do Sistema de Fracionamento GELFREE 8100, que partições amostras de proteínas complexas com base no peso molecular e recupera as frações em fase líquida. O vídeo descreve como a tecnologia funciona, como ele é usado, e fornece dados resultantes, com análise de eletroforese em gel de poliacrilamida de homogenato de fígado bovino fracionado.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

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

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JoVE Journal

Biology

16.3K visualizações.  Protein Discovery, Inc.. O vídeo que acompanha descreve o uso do Sistema de Fracionamento GELFREE 8100, que partições amostras de proteínas complexas com base no peso molecular e recupera as frações em fase líquida. O vídeo descreve como a tecnologia funciona, como ele é usado, e fornece dados resultantes, com análise de eletroforese em gel de poliacrilamida de homogenato de fígado bovino fracionado.

Vídeo: 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.

Preparação e fracionamento de Xenopus laevis Egg Extractos

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

Biologia

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.

Fracionamento em Subcelulares situ de aderentes e não aderentes células de mamíferos

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.

Visualização de Caenorhabditis elegans Estruturas cutícula Usando o Dye lipofílicos 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 Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

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.

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

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

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.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

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 Fracionamento e Análise de mamíferos Translatomes em uma escala genômica

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.

Estudando a Proteína de Qualidade Sistema de Controle de<em&gt; D. discoideum</em&gt; Usando temperatura controlada Imagens ao vivo celular

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.

Fraccionamento subcelular para activação de ERK tratamento de peptídeo derivado mitocondrial

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.

Gabinete de grafeno de células mamíferas quimicamente fixas para microscopia eletrônica 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 ...