Os autores agradecem ao Instituto Médico Howard Hughes para uma ciência UF para a concessão de Vida para AH

1. Preparando os cassetes <ol><li> Frente e verso limpo placas de um Nupage Invitrogen usado Novex cassete minigel, ou cassete minigel outro comercial. </li><li> Preparar ml aproximadamente 1-3 de epóxi de plástico e misturar bem de acordo com as instruções do fabricante. </li><li> Aplicar o epoxi para o bordo interior da placa frontal, onde as duas placas irá entrar em contacto quando a cassete é montado. </li><li> Deslizar um pedaço de papel de filtro ou de papelão através da fenda inferior da placa traseira, quando a montagem da cassete para evitar excesso de epóxi de selagem da abertura. Remover o papel de filtro, logo que as duas placas são montados. </li><li> Coloque grampos da pasta ao longo das bordas para uma perfeita vedação, e deixar que as cassetes sentar-se durante a noite para o epóxi para curar completamente. </li><li> Selar a fenda na parte inferior da placa traseira com fita isolante. </li></ol> 2. Verter o gel resolver <ol><li> Para um frasco de 125 ml braço lado, adicione o seguinte:2,25 ml de Tris-Base de Dados de 4X Resolving gel tampão de pH 8,70, 3,50 ml de acrilamida 30,8% / bisacrilamida, 3,25 ml de Tipo I de água grau reagente (GIR-H2O) para um volume total de 9 ml. </li><li> De-gás a solução durante 10 min, anexando o braço lateral do balão a um aparelho de vácuo, e colocando uma rolha invertido sobre o topo do frasco. </li><li> Transferir a 9 ml de isentos de gases solução para um tubo de centrífuga de 50 ml descartável de polipropileno e adicionar 30 uL de persulfato de ammounium 10% (EPA) e 6 uL de N, N, N &#39;, N&#39;, N-tetrametiletilenodiamina (TEMED) e misturar bem agitando a solução para evitar a introdução de bolhas. </li><li> Imediatamente despejar a solução na cassete, permitindo que a correr para baixo do lado da cassete para evitar quaisquer bolhas de formar entre as placas de gel. Encher a cassete para cerca de 2 cm a partir da borda superior da placa (mais curto) da frente. </li><li> Suavemente adicionar 0,5 ml de 1-butanol saturado com 1X tampão de gel de resolução e deixar que o gel polimerizar durante 1horas. </li></ol> 3. Verter o gel de empilhamento <ol><li> Adicionar o seguinte para um balão de 125 ml braço lateral: 0,75 ml de 4X tampão Tris pH-empilhamento de bases de gel de 6,80, 0,40 ml de acrilamida 30,8% / bisacrilamida, 1,85 ml de O 2 IRG-H para um volume total de 3 ml. </li><li> De-gás a solução durante 10 min, como descrito no passo 2. </li><li> Enquanto a solução de gel de empilhamento é desgaseificação, despeje o 1-butanol saturado com 1X tampão de gel de Resolving a partir do topo do gel de resolução. Lavar o topo do gel resolver uma vez com O. 2-H IRG Decantar a água e usar um papel absorvente ou filtro Whatman para absorver qualquer líquido remanescente. </li><li> Remover 3 ml da sua solução desgaseif içada e adicionar 9 ul de APS 10% e 2 ul de TEMED e misturar bem. </li><li> Rapidamente despejar a solução na cassete, permitindo que a correr para baixo do lado da cassete até que seja perto da borda superior da placa frontal curta de gel. </li><li> Inserir um pente gel limpo no topo da cassete de gel,colocar um clipe de ligante sobre a cassete sobre o pente para manter no lugar. Deixe a gel de empilhamento polimerizar durante 1 hora para durante a noite. </li></ol> 4. Armazenar o gel <ol><li> Quando o gel foi completamente polimerizado, remover os grampos da pasta e colocar a cassete dentro de uma bolsa de calor-selável, plástico ou um saco de plástico re-selável. </li><li> Introduzir 20 ml de 1X tampão de gel de empilhamento que contém azida de sódio a 0,1% para a bolsa. </li><li> Sele a bolsa por 5 segundos e verifique se não existem vazamentos no saco. </li><li> Armazenar o gel a 4 ° C. </li></ol> 5. Preparação das amostras <ol><li> Num tubo de microcentrífuga de paredes finas, adicionar até 6,5 uL de amostra de proteína, 7,5 ul de tampão de amostra 2x Novex Tris-Glicina de SDS, 1 ul de 100 mM de ditiotreitol (DTT) e IRG-H2O (se necessário) para uma volume total de 15 uL. </li><li> Misturar a amostra em vórtice, e brevemente centrifugar a amostra para trazer o conteúdo para o fundo do tubo. </li> &lt;li&gt; desnaturar a amostra a 70 ° C durante 10 min. </li></ol> 6. Executando o gel <ol><li> Configure o aparelho de gel Novex. </li><li> Adicionar 200 ml de Tris-Glicina tampão de corrida que contém 500 uL de antioxidante Nupage para a câmara interior, e 300 ml de Tris-Glicina O tampão de corrida para a câmara exterior. </li><li> Carregar as amostras de proteína desnaturadas (15 uL) para os poços, utilizando uma carga de gel ponta da pipeta. </li><li> Executar o gel a 200 V durante 60-70 min. </li></ol> 7. A coloração do gel <ol><li> Remova a cassete do aparelho de gel e quebrar o selo epóxi por erguer as duas placas de gel de plástico separados utilizando uma faca de gel ou de uma espátula rígida. Cuidadosamente separar o gel a partir das placas, tendo o cuidado de não rasgar o gel. </li><li> Lavar o gel com IRG-H2O três vezes com agitação suave. </li><li> Corar o gel com Invitrogen SimplyBlue SafeStain durante 1 hora com agitação suave. </li><li> Decantar a solução de coloração edestain o gel com O. 2-H IRG </li></ol> 8. Limpando as gavetas <ol><li> Use uma espátula de metal para raspar ou descascar todo o epóxi sobre as cassetes para que possam ser reutilizadas. </li><li> Lava-se cada uma das placas de gel de plástico com uma solução de detergente suave, e lavar bem com água da torneira seguida por água destilada. </li></ol> 9. Os resultados representativos Devido epoxi irá formar uma vedação estanque a água, (Figura 1), as cassetes não irá vazar quando a solução é vertida em acrilamida-los. Além disso, as cassetes de gel pode ser facilmente aberto para recuperar o gel para a coloração, eo epóxi pode ser retirada das cassetes para permitir a sua continuação reutilização. Como mostrado na Figura 2, o gel mostra nítida separação de marcadores proteicos e bandas amostra adequada para a electroforese de proteínas de rotina. <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="Figura 1" /> Figura 1. Aplicaçãocatião de epoxi para selar cassetes minigel. Uma fina camada de epóxi é aplicada à extremidade de uma das cassetes antes da montagem. Para realçar o epóxi, que era falso-cor vermelha nesta imagem. <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="A Figura 2" /> Figura 2. Separação de proteínas sobre um gel de SDS-Tris-glicina vertida usando re-utilizados cassetes minigel. SDS-PAGE foi realizada como descrito acima. Após electroforese o gel foi removido do cassete minigel, coradas em SimplyBlue SafeStain como por instruções do fabricante, e fotografados utilizando luz transmitida branco. Pista M contém SeeBlue Plus2 padrões pré-corados de proteína. Os pesos moleculares das proteínas em pista M são indicados no lado direito do gel em quilodaltons (kDa). As outras pistas mostram as proteínas a partir de um lisado limpo a partir de bactérias que expressam uma glutationa-S-transferase de proteínas (GST)-etiquetado. As proteínas são indicados os lavada a partir do ggrânulos lutathione antes da eluição da proteína GST-etiquetado.

<ol>
	<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Raymond, S., Weintraub, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide+gel+as+a+supporting+medium+for+zone+electrophoresis.">Acrylamide gel as a supporting medium for zone electrophoresis.</a> Science. 130, 711-711 (1959).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem. Biophys. Res. Commun. 28, 815-820 (1967).</li></ol>

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Eletroforese em gel de proteína é um método indispensável para laboratórios de biologia molecular mais. Minigels comercialmente disponíveis fornecer um elevado nível de conveniência, mas pode ser caro. Aqui nós mostramos como reutilizar as fitas minigel de uma popular marca de minigels disponíveis comercialmente. Este método retém a conveniência de ter uma fonte pronta de géis, mas a uma fracção do custo de minigels comercialmente disponíveis. Um passo chave neste processo está a aplicar a quantidade c...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Invitrogen Nupage Novex Cassete minigel (1,0 mm) </td><td> Invitrogen </td><td> NC2010 </td><td> Disponível em 2, 5, 10, 12, 15 e os poços 2D </td></tr><tr><td> Epoxy plástico </td><td> Loctite </td><td> 108771 </td><td></td></tr><tr><td> Fita elétrica </td><td> Escocês </td><td></td><td></td></tr><tr><td> 125 mL frasco braço lateral </td><td> Pyrex </td><td> 5360 </td><td></td></tr><tr><td> 4X Tris-Base Resolving tampão de gel </td><td></td><td></td><td> 1,5 M pH 8,7 Tris-base </td></tr><tr><td> acrilamida </td><td> Fisher Scientific </td><td> 01065-500 </td><td></td></tr><tr><td> Bis-acrilamida </td><td> Biomedicals MP, Inc. </td><td> 800173 </td><td></td></tr><tr><td> Solução de acrilamida 30,8% </td><td></td><td></td><td> 30:0.8 acrilamida: bis-acrilamida em O 2-H IRG </td></tr><tr><td> APS 10% </td><td> Fisher Scientific </td><td> BP179-25 </td><td> 0,1 g de persulfato de amónio em 1 ml IRG-H2O, preparado imediatamente antes da utilização </td></tr><tr><td> Temed </td><td> Organics Arcos </td><td> 138450500 </td><td></td></tr><tr><td> 1-butanol saturado com 1X tampão de gel de Resolving </td><td></td><td></td><td> 05:02 1-butanol a 1X tampão de gel de Resolving </td></tr><tr><td> 4X Tris-Base de Dados de tampão de gel de empilhamento </td><td></td><td></td><td> 1,5 M pH 6,8 Tris-base </td></tr><tr><td> Pente Gel </td><td> Invitrogen </td><td> NC3015 </td><td> 15 assim, 1,0 mm de espessura </td></tr><tr><td> Sealer Impulse </td><td> TEW aparelhos eléctricos </td><td></td><td></td></tr><tr><td> Bolsa selável calor </td><td> Kapak / Scotchpak </td><td></td><td> 1 litro tamanho (6,5 &quot;x 8&quot;) </td></tr><tr><td> Gel de tampão de armazenamento </td><td></td><td></td><td> 1X tampão de gel de empilhamento com azida de sódio a 0,1% </td></tr><tr><td> Novex tampão de amostra SDS Tris-Glicina (2X) </td><td> Invitrogen </td><td> LC2676 </td><td></td></tr><tr><td> TDT </td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X tampão de corrida Tris-Gylcine </td><td> Invitrogen </td><td> LC2675 </td><td> 1L de estoque 10X: 29,0 g Tris-Base, 144,0 g Glicina, 10,0 g SDS </td></tr><tr><td> XCell SureLock Mini-Cell </td><td> Invitrogen </td><td> EI0001 </td><td></td></tr><tr><td> Faca Gel </td><td> Invitrogen </td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue Seguro Mancha </td><td> Invitrogen </td><td> LC6065 </td&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

A avaliação de proteínas utilizando dodecil sulfato de sódio em gel de poliacrilamida a análise de electroforese (SDS-PAGE) é uma técnica comum utilizado por pesquisadores Bioquímica e Biologia Molecular 1-4. Para laboratórios que realizam análises diárias de proteínas, o custo de géis de poliacrilamida comercialmente disponível (~ $ 10/gel) pode ser considerável ao longo do tempo. Para reduzir este custo, alguns pesquisadores preparar seus próprios géis de poliacrilamida. Os métodos tradicionais de verter estes geles tipicamente utilizar equipamento especializado e placas de vidro de gel que podem ser caros e impede vertendo géis muitos e armazená-los para uso futuro. Além disso, a manipulação de placas de vidro durante a limpeza ou gel vertendo pode resultar em quebra acidental criar um risco de segurança, que pode impedir o seu uso em classes de laboratório de graduação. Nosso protocolo demonstra como colocar o gel de proteínas simultaneamente pela reciclagem Invitrogen Nupage cassetes Novex minigel, e ma baratariais comprado em uma loja da melhoria home. Este método económico e simplificado inclui uma forma de armazenar os géis a 4 ° C durante algumas semanas. Ao re-utilizando as cassetes de gel de plástico de géis disponíveis no mercado, laboratórios que funcionam géis de proteína freqüentes podem economizar custos significativos e ajudar o meio ambiente. Além disso, cassetes de gel de plástico são extremamente resistentes à quebra, o que os torna ideal para salas de aula de laboratório de graduação.

Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Pouring and Running a Protein Gel by reusing Commercial Cassettes

Nosso protocolo demonstra como colocar o gel de proteínas múltiplas em um momento de reciclagem Invitrogen Nupage cassetes Novex minigel e materiais baratos comprados em uma loja da melhoria home. Este método económico e simplificado inclui uma forma de armazenar os géis a 4 ° C durante algumas semanas. Ao re-utilizando as cassetes de gel de plástico de géis disponíveis no mercado, laboratórios que funcionam géis de proteína freqüentes podem economizar custos significativos e ajudar o meio ambiente.

Derramando e executando um gel de proteína através da reutilização Cassetes comerciais

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

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Research

JoVE Journal

Biology

24.8K visualizações.  University of Florida. Nosso protocolo demonstra como colocar o gel de proteínas múltiplas em um momento de reciclagem Invitrogen Nupage cassetes Novex minigel e materiais baratos comprados em uma loja da melhoria home. Este método económico e simplificado inclui uma forma de armazenar os géis a 4 ° C durante algumas semanas. Ao re-utilizando as cassetes de gel de plástico de géis disponíveis no mercado, laboratórios que funcionam géis de proteína freqüentes podem economizar custos...

Vídeo: Pouring and Running a Protein Gel by reusing Commercial Cassettes

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Triagem para Agregação de amilóide por Semi-desnaturação Eletroforese Gel Detergente-Agarose

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Biologia

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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 ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Melhorar a taxa de sucesso de cristalização de proteínas pela Random Microseed Matrix Screening

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interações proteína-proteína visualizado por Bimolecular Fluorescência de Complementação em tabaco Protoplastos e folhas

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Detecção de glicosaminoglicanos por Electrophoresis gel poliacrilamida e coloração de prata

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Um modelo de roda de corrida simples e barato para treinamento de resistência progressiva em camundongos

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Simplificando os estudos de angiogênese com um ensaio de tampão de gel de matriz modificada

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Autor em destaque: Investigando a angiogênese e a permeabilidade dos vasos por meio de um ensaio de tampão de gel de matriz modificada

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Ensaio de plugue de gel de matriz in vivo modificado para estudos de angiogênese

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...