Este estudio fue financiado por: Red Nacional de Investigación sobre el Envejecimiento (NFN S93) por la Fundación de Ciencias de Austria (FMF), Comisión Europea Integrada de Proyectos y MiMAGE PROTEOMAGE, Países Bajos Genómica Iniciativa / Organización Neerlandesa para la Investigación Científica (IGN / NWO, 05040202 y 050 - 060-810 NCHA), financiado por la UE Red de Excelencia Duración (6 º PM 036.894) y el Programa de Innovación Tecnológica Orientada sobre Genómica (SenterNovem; IGE01014 y IGE5007).

1. Construcción de plásmidos <ol><li> Orden de ratón oligo-nucleótidos que codifica para dgn (ACKNWFSSLSHFVIHL 11) y para dgnFS (HARTGSLACPTSSSICE) y ligarlo en el vector pEGFP-C1 para obtener la fusión de la GFP con dgn / dgnFS (Figura 1). </li><li> Amplificar la secuencia codificante de GFP y GFP-dgn dgnFS-por PCR de acuerdo con el protocolo del kit de clonación pENTR Directional TOPO y continuar con la pLenti6/V5 direccional TOPO Cloning Kit (Figura 6). </li></ol> 2. Virus Producción <ol><li> El día antes de la transfección (Día 1) semilla fuera HEK 293FT células en unos frascos T75 para que puedan ser 90-95% de confluencia en el día de la transfección. </li><li> En el día de la transfección diluir 36 l de Lipofectamine 2000 reactivo en 1,5 ml de DMEM sin suero y antibióticos en un falcon de 15 ml. Utilice otro Falcon de 15 ml y diluir 3 mg pLenti6/V5 que lleva el ADN complementario, ya sea para la construcción de buenas prácticas agrarias<html> -Dgn o GFP-dgnFS, 2,5 g envolvente pMD2.G plásmido que codifica y 7,5 g psPAX2 esqueleto del vector en 1,5 ml de DMEM sin suero y antibióticos. Después de 5 min el ADN diluido se combina con la solución diluida de reactivos Lipofectamine 2000. </li><li> Incubar la mezcla durante 20 min a temperatura ambiente para permitir que los complejos de ADN-lipofectamina a forma. </li><li> Retire el medio de las células HEK 293FT, reemplácelo con cuidado por 7 ml de medio (sin antibióticos) y se añade la mezcla de ADN-lipofectamina al matraz y roca hacia adelante y atrás para mezclar (Día 2). Incubar el frasco durante la noche a 37 ° C en un humidificado 5% de CO 2 incubadora. </li><li> Cambio de medio el día siguiente (10 ml de medio sin antibióticos; Día 3). </li><li> Después de 48 horas (día 5) cosecha sobrenadante, centrifugar a 300 xg durante 5 min a temperatura ambiente y se filtra el sobrenadante a través de un filtro de 0,45 micras PVDF. </li></ol> Medio de cultivo - DMEM (HEK 293FT) tienda de campaña &quot;&gt; DMEM FBS 10% 0,1 mM MEM NEAA 6 mM de L-glutamina 1 mM de piruvato sódico MEM 1% de Pen-Strep (opcional) 500 g / ml de geneticina (opcional) 3. Virus Concentración por polietilenglicol (PEG) Precipitación <ol><li> Añadir una solución de glicol de polietileno volumen (50 Polietilenglicol mM, 41 mM NaCl, autoclave, pH = 7,2; PEG) a cuatro volúmenes de sobrenadante y se incuba durante 2 horas a 4 ° C. Con cuidado, se mezcla cada 20-30 minutos por inversión. </li><li> Centrifugue a 1500 xg durante 30 min a 4 ° C. Una bola blanca debe ser visible. </li><li> Aspirar el sobrenadante y se centrifuga de nuevo a 1500 xg durante 5 min a 4 ° C. Aspirar la solución de PEG restante. </li><li> Resuspender el sedimento en medio o tampón fosfato salino (PBS) pipeteando arriba y abajo y vigorosamente vórtice durante 20 a 30 segundos. Como una guía para utilizar 500 l un matraz T75 y alícuota a 100 l. Almacenar el virus a -80 ° C. </li></ol><ol><li> Semilla fuera 5 × 10 4 U2-OS células a cada pocillo de una placa de 6-y el día antes de la transfección. </li><li> En el día de la transfección eliminar el medio de cultivo y sustituirla por 1 ml de DMEM que contenía 8 g / ml de polibreno (bromuro de hexadimetrina). Diluir el virus concentrado sobrenadante 1:10 y 1:100 y añadir 1 l cada uno a un pocillo. Para concentraciones más altas de virus usan 1 l, l 5 y 10 l de sobrenadante de virus concentrada para los pocillos restantes. Uno bien se deja como un control positivo. </li><li> El día siguiente sustituir el medio por 2 ml de DMEM. </li><li> Comienza con la selección del día siguiente. Aplicar 10 g / ml blasticidina en cada pocillo. Sustituya el medio que contiene antibiótico cada dos días. </li><li> Aproximadamente 6-7 días después de comenzar la selección de las células no transducidas están muertos. Para la tinción de lavar las células tres veces con PBS y se cubren las células con violeta cristal (10 mg / l de violeta cristalen 20% de etanol) y se incuba durante 5-10 min a temperatura ambiente. Aspirado de cristal violeta (puede ser reutilizado varias veces) y enjuagar los pocillos dos veces con ddH 2 O y secar la placa. </li><li> Contar las colonias y se multiplican con 1.000 y la dilución correspondiente (Figura 7). Este procedimiento permite calcular las unidades de transfección (TU) del virus / ml. </li></ol> Medio de cultivo - DMEM (HFF-2/U2-OS) DMEM FBS 10% 6 mM L-glutamina 1% de Pen-Strep 5. Transducción de fibroblastos diploides humanos (Este procedimiento puede ser utilizado para cualquier tipo de célula.) <ol><li> Semilla fuera 5 × 10 4 fibroblastos diploides humanas (HDF) a 6-y las placas el día antes de la transducción. </li><li> Utilice una multiplicidad de infección de dos junto con 8 mg / ml de polibreno como potenciador de la transducción en un total de un mililitro. </li><li> Cambio de medio al día siguiente. </li><li> ¿Cuándolas células están en un 70-80% de confluencia comienzo de la selección con 10 mg / ml de blasticidina. Después de completada la selección (sin células no transfectadas mueren más) la expansión de las células puede iniciar y las células están listas para experimentos. Selección crónica con blasticidina permite generar una línea celular estable que sobreexpresan GFP-dgn o GFP-dgnFS proteínas de fusión, listos para medir la actividad del proteasoma. Este procedimiento garantiza una eficacia de transfección muy alta independiente del tipo de células. </li></ol> 6. Medición por citometría de flujo <ol><li> Semilla out 1 × 10 5 células (ya sea en libros o GFP-dgn dgnFS GFP-) el día antes del experimento. </li><li> Tratar las células de control de 3 horas antes de la medición con el inhibidor del proteasoma, por ejemplo, 100 mg / ml LLnL. </li><li> Lavar dos veces con PBS y se cosechan las células usando 0,5 ml de tripsina. Para detener la tripsinización utilizar 4,5 ml de medio de cultivo y transferir las células a un Falcon de 15 ml. </li><li> Girar tél las células a 300 xg durante 5 min a temperatura ambiente. </li><li> Aspirar el medio, resuspender las células en PBS y centrifugar de nuevo a 300 xg durante 5 min a 37 ° C. </li><li> Aspirar el PBS, resuspender en 400 l de tampón FACS (10 mM Hepes, 140 mM NaCl, 2,5 mM CaCl 2, pH = 7,4) y transferir las células a tubos de FACS. </li><li> Mantener las células en muestras de hielo y medida por citometría de flujo. Las células no se deben almacenar más de 30 min a 4 ° C. </li></ol> 7. Los resultados representativos La proteína de fusión GFP-dgn lleva una secuencia que se dirige al proteosoma y por lo tanto la proteína es degradada inmediatamente, sino que corresponde a la disminución de la señal de fluorescencia GFP. El mutante de desplazamiento de marco (GFP-dgnFS) lleva una versión mutada de esta secuencia y no es degradado por la proteasoma, sino que conduce a la mayor fluorescencia verde. Por estas razones, joven HDF con la actividad del proteasoma espera alta y transducidas con GFP-dgn muestran una baja (6% de células positivas) señal de fluorescencia en tanto la medición de citometría de flujo y en epifluorescencia (Figura 2A y B, Figura 3). Lo mismo HDF transduced con las buenas prácticas agrarias-dgnFS mostrar el 39,7% de las células positivas. El tratamiento de las células con inhibidor del proteasoma LLnL (N-acetil-L-leucil-L-leucil-L-norleucinal) planteó la señal hasta un máximo de 62,9% en ambos, GFP-dgn y GFP-dgnFS células (Figura 2A y B ). Para determinar la posible disminución de la actividad del proteasoma en edades comprendidas entre muestras de piel humana, los fibroblastos dérmicos aislados de donantes jóvenes, de mediana edad y ancianos fueron infectadas con GFP y GFP-dgn dgnFS, como se ha descrito anteriormente y se cultiva en el número mismo pasaje antes del análisis por flujo citometría. En estos experimentos, un aumento claro en la señal de GFP entre individuos jóvenes (11,2 ± 0,88% células GFP-positivas) y de mediana edad donantes (20,4 ± 2,27% de opiniones positivas GFP-células, p = 0,003) se ha observado, lo que indica una disminuir en proteasome actividad en las muestras obtenidas de donantes de edad 7 (Figura 4). No mayor disminución en la actividad del proteasoma se observó en fibroblastos aislados de individuos de más edad (Figura 4). La intensidad de fluorescencia de GFP-dgnFS era en todos los casos ~ 90% (datos no mostrados) lo que indica una alta eficiencia de transfección de los tres diferentes grupos de edad. <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="Figura 1" /> Figura 1. La secuencia GFP y GFP-dgn dgnFS-. Muestra es el extremo 3 &#39;de GFP (verde) el sitio de clonación múltiple del vector pEGFP-CL1 (gris) y la secuencia de dgn / dgnFS (rojo). <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="Figura 2" /> Figura 2. Análisis de citometría de flujo de las buenas prácticas agrarias y las buenas prácticas agrarias-dgn dgnFS fibroblastos diploides humanos. A. fibroblastos jóvenes de prepucio humano (HFF-2) fueron infectadas con vectores lentivirales que transportan una proteína fluorescente verde (GFP)-dgn gen o un dgnFS GFP-(frameshift) construir, como se indica y se analizaron para la fluorescencia de GFP mediante citometría de flujo (FACS Canto II, Becton Dickinson). Donde se indique, las células se trataron también durante 3 h con inhibidor del proteasoma N-acetil-L-leucil-L-leucil-L-norleucinal (LLNL). Las células no infectadas se utilizaron como control. Los experimentos se realizaron por triplicado. B. Los datos son representativos de tres experimentos independientes. Los números reflejan la cantidad de células GFP positivas. <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="Figura 3" /> Figura 3. Análisis de microscopía de fluorescencia de GFP y GFP-dgn-dgnFS células HFF-2. Joven HFF-2 fueron tratados como en la Figura 2. A los 9 días después de la infección, las células fueron visualized por fluorescencia y microscopía de contraste de fase. <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="Figura 4" /> Figura 4. Los cambios en la actividad del proteasoma en el envejecimiento de la piel humana. Los fibroblastos de prepucio humano de nueve donantes diferentes en los grupos de edad indicados se expandieron mínimamente y se infectaron con constructos de lentivirus que codifican para GFP-DGN. A los 9 días después de la infección, las células fueron analizadas por citometría de flujo. Los datos se obtuvieron en tres muestras por grupo de edad por duplicado (± SE). <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="Figura 5" /> Figura 5. Enfoque experimental. Custom-oligo nucleótidos para dgn y dgnFS se clonan en el vector pEGFP-C1 y los virus se producen para cada constructo utilizando células HEK 293FT. El título de los virus se determina. Las células son transducidas con el virus y expandido. Después de que el trato de favor que las células se analizanpara la señal de fluorescencia por citometría de flujo. <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="La figura 6" /> Figura 6. Mapa de Plenti GFP-dgn. El mapa del pLenti6/V5-DEST vector que incluye la secuencia de GFP-dgn se muestra. Abreviaturas: pCMV (promotor CMV), GFP-dgn (secuencia de GFP-dgn), P SV40 (SV40 promotor temprano), EM7 (EM7 promotor), blasticidina (gen blasticidina resistencia), ΔU3 / LTR 3 &#39;(3&#39; LTR con eliminada región U3), pA SV40 (SV40 señal de poliadenilación), ampicilina (gen de resistencia a ampicilina), pUC ori (origen de pUC), PRSV / LTR en 5 &#39;(RSV / LTR 5&#39; promotor híbrido), Ψ (VIH-1 señal de empaquetamiento Ψ ), RRE (VIH-1 elemento de respuesta a Rev). <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="Figura 7" /> Figura 7. U2-OS placa de titulación. U2-OS se sembraron sobre una placa de 6 pocillos y se transfectaron con diferentes concentraciones de virus (dilución de 1/100 y 1/10, 1, 5 y 10 l de concentrated sobrenadante viral). Un pozo se utilizó como control no transfectadas (NT). Después de 6-7 días, las células se tiñeron con cristal violeta y se secaron al aire.

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No hay conflictos de interés declarado.

La primera publicación utilizando la proteína fluorescente verde (GFP) como sustrato reportero de ubiquitina-proteasoma actividad se publicó en 2000 12. Desde entonces, GFP se ha convertido en una herramienta común para visualizar las actividades celulares, especialmente el proceso de ubiquitina-proteasoma. Para supervisar la ubiquitina-proteasoma actividad in vivo de un modelo de ratón transgénico con un reportero GFP basada ha introducido 13. Adicional a la investigación ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> pEGFP-C1 Vector </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR Directional TOPO Cloning Kit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 direccional TOPO Cloning Kit </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine 2000 Reactivo </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> PVDF filtro (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polietileno glicol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Fosfato de Dulbecco tamponada Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> hexadimetrina bromuro </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidina </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> El cristal violeta </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS tubos </td><td> BD Biosciences </td><td></td></tr><tr><td> La penicilina estreptomicina (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutamina 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Suero bovino fetal (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM no aminoácidos esenciales (AANE) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM piruvato sódico 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-glucosa (45%) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Geneticina </td><td> Invitrogen </td><td> 11811023 </td></tr><tr><td> CaCl2 </td><td> Merck </td><td> C5080 </td></tr><tr><td> Hepes </td><td> Sigma </td><td> H3375 </td></tr><tr><td> Tripsina-EDTA (0,05%) </td><td> Invitrogen </td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

Proteasoma es el principal organelo intracelular implicada en la degradación proteolítica de proteínas anormales, mal dobladas, dañadas u oxidadas 1, 2. El mantenimiento de la actividad del proteasoma fue implicado en muchos procesos celulares clave, como respuesta al estrés celular 3, la regulación del ciclo celular y la diferenciación celular 4 o en la respuesta del sistema inmune 5. La disfunción del sistema ubiquitina-proteasoma se ha relacionado con el desarrollo de tumores y enfermedades neurodegenerativas 4, 6. Además, una disminución en la actividad del proteasoma se encontró como una característica de la senescencia celular y el envejecimiento organismal 7, 8, 9, 10. A continuación, se presenta un método para medir la actividad ubiquitina-proteasoma en células vivas utilizando una proteína de fusión GFP-dgn. Para ser capaz de controlar la actividad de la ubiquitina-proteasoma en células vivas primarias, ADN complementario construye codifica para una proteína fluorescente verde (GFP)-dgn proteína de fusión (GFP-dgn, inestable) y una variante con una mutación de desplazamiento de marco (GFP-dgnFS, estable 11) se insertan en vectores de expresión lentiviral. Se prefiere esta técnica sobre técnicas de transfección tradicionales, ya que garantiza una eficacia de transfección muy alta independiente del tipo de célula o de la edad del donante. La diferencia entre la fluorescencia mostrada por el GFP-dgnFS (estable) de proteína y la proteína de desestabilizado (GFP-dgn) en ausencia o presencia de inhibidor del proteasoma puede ser utilizado para estimar la ubiquitina-proteasoma actividad en cada cepa de célula particular. Estas diferencias pueden ser supervisado por microscopía de epifluorescencia o puede ser medido por citometría de flujo.

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Un método para supervisar la actividad de ubiquitina-proteasoma en células vivas se describe. Un degron-GFP desestabilizado-(GFP-dgn) y una estable GFP-dgnFS proteína de fusión se generan y se transduce en la célula usando un vector de expresión lentiviral. Esta técnica permite generar un GFP-dgn/GFP-dgnFS estable línea celular que expresa en la que se puede ubiquitina-proteasoma actividad fácilmente evaluada utilizando epifluorescencia o citometría de flujo.

Control de la ubiquitina-proteasoma actividad en las células vivas utilizando un degron (dgn)-desestabilizado la proteína verde fluorescente (GFP)-basada en proteína indicadora

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

monitoring-ubiquitin-proteasome-activity-living-cells-using-degron

Research

JoVE Journal

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

16.6K vistas.  Institute for Biomedical Aging Research. Un método para supervisar la actividad de ubiquitina-proteasoma en células vivas se describe. Un degron-GFP desestabilizado-(GFP-dgn) y una estable GFP-dgnFS proteína de fusión se generan y se transduce en la célula usando un vector de expresión lentiviral. Esta técnica permite generar un GFP-dgn/GFP-dgnFS estable línea celular que expresa en la que se puede ubiquitina-proteasoma actividad fácilmente evaluada utilizando epifluorescencia o citometría de flujo.

Video: Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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

Investigación

Biología

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.

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 ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolecular complementación de fluorescencia (BiFC) Ensayo de interacción proteína-proteína en las células de cebolla con la pistola de genes Helios

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching ensayos (FRAP y FLIP) para medir la dinámica de la cromatina de proteínas en células vivas madre embrionarias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Ensayo de crecimiento basado en reportero para el Análisis Sistemático de la degradación de proteínas

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genoma toda la Proteína-proteína interacción Screening por Complementación de ensayo de proteínas-fragmento (PCA) en las células vivas

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

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.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<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 ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Un protocolo para la evaluación funcional de la Proteína Total-mutagénesis de saturación Bibliotecas La utilización de secuenciación de alto rendimiento

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...