Cette étude a été financée par le Réseau national de recherche sur le vieillissement (PNN S93) par le scientifique autrichien Foundation (FWF), la Commission européenne des projets intégrés et MiMAGE PROTEOMAGE, Pays-Bas Genomics Initiative / Organisation néerlandaise pour la recherche scientifique (NGI / NWO, 05040202 et 050 - 060-810 LPN), financé par l&#39;UE réseau d&#39;excellence Durée de vie (FP6 036894) et le Programme de recherche et d&#39;innovation orientée sur la génomique (SenterNovem; IGE01014 et IGE5007).

1. Construction des plasmides <ol><li> Afin personnalisé oligo-nucléotides codant pour dgn (ACKNWFSSLSHFVIHL 11) et pour dgnFS (HARTGSLACPTSSSICE) et le ligaturer dans le vecteur pEGFP-C1 afin d&#39;obtenir la fusion de la GFP avec dgn / dgnFS (figure 1). </li><li> Amplifier la séquence codant pour la GFP-dgn et GFP-dgnFS par PCR selon le protocole du kit de clonage TOPO directionnel pENTR et continuer avec le kit TOPO directionnel pLenti6/V5 clonage (figure 6). </li></ol> 2. Production de virus <ol><li> Le jour avant la transfection (Jour 1) de graines sur les cellules HEK 293FT dans un des flacons T75 de sorte qu&#39;ils seront confluentes à 90-95% le jour de la transfection. </li><li> Le jour de la transfection diluer 36 ul de lipofectamine 2000 réactif dans 1,5 ml de DMEM sans sérum et d&#39;antibiotiques dans un falcon de 15 ml. Utilisez un autre Falcon de 15 ml et diluer 3 pg pLenti6/V5 portant l&#39;ADN complémentaire de construire soit pour la GFP<html> -Dgn ou GFP-dgnFS, 2,5 mg pMD2.G encodage plasmide d&#39;enveloppe et de 7,5 mg psPAX2 squelette du vecteur dans 1,5 ml de DMEM sans sérum et d&#39;antibiotiques. Après 5 min de l&#39;ADN dilué est combiné avec le réactif dilué lipofectamine 2000. </li><li> Incuber le mélange pendant 20 min à température ambiante pour permettre les complexes ADN-lipofectamine à la forme. </li><li> Retirez le support de cellules HEK 293FT, remplacez-le avec soin par 7 ml de milieu (sans antibiotiques) et ajouter le mélange d&#39;ADN-lipofectamine dans le ballon et it rock d&#39;avant en arrière pour le mélange (Jour 2). Incuber le ballon pendant une nuit à 37 ° C dans une atmosphère humidifiée à 5% de CO 2 incubateur. </li><li> Changer le milieu le lendemain (10 ml de milieu sans antibiotiques; Jour 3). </li><li> Après 48 heures (jour 5) surnageant récolte, centrifuger à 300 xg pendant 5 min à température ambiante et filtrer le surnageant à travers un filtre de 0,45 um PVDF. </li></ol> Milieu de culture - DMEM (HEK 293FT) tente &quot;&gt; DMEM FBS 10% 0,1 mM MEM NEAA 6 mM de L-glutamine Pyruvate de sodium 1 mM MEM 1% Pen-Strep (facultatif) 500 pg / ml de généticine (facultatif) 3. Concentration du virus par le polyéthylène glycol (PEG) Précipitations <ol><li> Ajoutez une solution de glycol polyéthylène de volume (50 Polyéthylène glycol mM, 41 mM de NaCl, autoclave, pH = 7,2; PEG) à quatre volumes de surnageant et laisser incuber pendant 2 heures à 4 ° C. Mélanger soigneusement à chaque 20-30 minutes en retournant. </li><li> Centrifuger à 1500 xg pendant 30 min à 4 ° C. Une pastille blanche doit être visible. </li><li> Aspirer le surnageant et centrifuger à nouveau à 1500 xg pendant 5 min à 4 ° C. Aspirer la solution de PEG restant. </li><li> Remettre en suspension le culot dans du milieu ou tampon phosphate salin (PBS) par pipetage de haut en bas et vigoureusement au vortex pendant 20 à 30 secondes. A titre indicatif utiliser 500 ul pour un flacon T75 et il aliquote de 100 ul. Stocker le virus à -80 ° C. </li></ol><ol><li> Graines à 5 × 10 4 cellules U2-OS à chaque puits d&#39;une plaque à 6 puits le jour avant la transfection. </li><li> Le jour de la transfection enlever le milieu de culture et le remplacer par 1 ml de DMEM contenant 8 pg / ml de polybrène (hexadiméthrine bromure). Diluer le concentré de virus surnageant 1:10 et 1:100 et ajouter 1 ul de chacune à un puits. Pour des concentrations plus élevées de virus utilisent 1 pi, 5 pi et 10 pi de surnageant de virus concentré pour les puits restants. Un puits est laissé comme témoin positif. </li><li> Le jour suivant, remplacer le milieu par 2 ml de DMEM. </li><li> Début de la sélection le lendemain. Appliquer 10 pg / ml blasticidine sur chaque puits. Remplacez le support antibiotique contenant tous les deux jours. </li><li> Environ 6-7 jours après le début de la sélection des cellules untransduced sont morts. Pour la coloration des cellules laver trois fois avec du PBS et les cellules couvrir au cristal violet (10 mg / l de cristal violet dedans de l&#39;éthanol 20%) et incuber pendant 5-10 min à température ambiante. Aspirer cristal violet (peut être réutilisé plusieurs fois) et rincer les puits deux fois avec ddH 2 O et sécher la plaque. </li><li> Compter les colonies et se multiplient à 1000 et la dilution correspondante (figure 7). Cette procédure permet de calculer les unités de transfection (TU) du virus / ml. </li></ol> Milieu de culture - DMEM (HFF-2/U2-OS) DMEM FBS 10% 6 mM de L-glutamine 1% Pen-Strep 5. La transduction des fibroblastes diploïdes humains (Cette procédure peut être utilisée pour n&#39;importe quel type de cellule.) <ol><li> Graines à 5 × 10 4 fibroblastes diploïdes humains (HDFS) à des plaques 6 puits la veille de transduction. </li><li> Utiliser une multiplicité d&#39;infection de deux avec 8 ug / ml de polybrène en tant que renforçateur de transduction dans un total de un millilitre. </li><li> Changer le milieu de la journée suivante. </li><li> Quandles cellules sont à 70-80% de confluence début de la sélection avec 10 ug / ml blasticidine. Une fois la sélection terminée (pas de cellules non transfectées meurent plus) l&#39;expansion des cellules peut commencer et les cellules sont prêtes pour des expériences. Sélection chronique avec blasticidine permet de générer une lignée cellulaire stable surexprimant GFP-dgn ou protéines de fusion GFP-dgnFS, prêt à mesurer l&#39;activité du protéasome. Ce procédé garantit une efficacité de transfection très élevée indépendante du type cellulaire. </li></ol> 6. Mesure par cytométrie en flux <ol><li> Graines à 1 × 10 5 cellules (portant soit GFP-dgn ou GFP-dgnFS) le jour avant l&#39;expérience. </li><li> Traiter les cellules de contrôle 3 heures avant la mesure avec l&#39;inhibiteur de protéasome, par exemple 100 pg / ml LLnL. </li><li> Laver deux fois avec du PBS et récolter les cellules en utilisant 0,5 ml de trypsine. Pour arrêter la trypsinisation utiliser 4,5 ml de milieu de culture et de transférer les cellules à un faucon 15 ml. </li><li> Spin til cellules à 300 xg pendant 5 min à température ambiante. </li><li> Aspirer le milieu, remettre en suspension les cellules dans du PBS et centrifuger à nouveau à 300 xg pendant 5 minutes à 37 ° C. </li><li> Aspirer le PBS, remettre en suspension dans 400 ul de tampon FACS (HEPES 10 mM, NaCl 140 mM, 2,5 mM de CaCl2, pH = 7,4) et de transférer les cellules dans des tubes FACS. </li><li> Gardez les cellules sur des échantillons de glace et de mesure par cytométrie en flux. Les cellules ne doivent pas être stockés plus de 30 min à 4 ° C. </li></ol> 7. Les résultats représentatifs La protéine de fusion GFP-dgn porte une séquence qui est destiné à le protéasome et donc la protéine est immédiatement dégradée, elle correspond à la diminution de la fluorescence de la GFP signal. Le cadre de lecture (GFP-dgnFS) mutant porteur d&#39;une version mutée de cette séquence et n&#39;est pas dégradé par le protéasome, elle conduit à l&#39;augmentation de fluorescence verte. Pour ces raisons, le jeune HDFS les contraintes auxquelles s&#39;attend l&#39;activité du protéasome élevé et transduites avec GFP-dgn montrent une faible (6% de cellules positives) signal de fluorescence à la fois la mesure par cytométrie en flux et en épifluorescence (figure 2A et B, Figure 3). Le même HDFS transduites avec GFP-dgnFS afficher 39,7% de cellules positives. Le traitement des cellules avec un inhibiteur de protéasome LLnL (N-acétyl-L-leucyl-L-leucyl-L-norleucinal) a soulevé le signal à concurrence de 62,9% dans les deux, la GFP et GFP-dgn dgnFS cellules (figure 2A et B ). Pour déterminer éventuelle baisse de l&#39;activité du protéasome dans vieillards échantillons de peau humaine, des fibroblastes dermiques isolés à partir de donneurs jeunes, d&#39;âge moyen et vieux ont été infectées par le GFP-dgn et GFP-dgnFS, tel que décrit ci-dessus et cultivées pour le nombre de passages même avant l&#39;analyse par les flux de cytométrie. Dans ces expériences, une augmentation nette du signal GFP entre les individus jeunes (11,2 ± 0,88% des cellules GFP-positives) et d&#39;âge moyen 20,4 ± bailleurs de fonds (2,27% cellules GFP-positives, p = 0,003) a été observée, ce qui indique une diminution de la proteasome activité dans des échantillons provenant de donneurs âgés de 7 (figure 4). Aucune nouvelle baisse de l&#39;activité du protéasome a été observée dans les fibroblastes isolés à partir de vieux individus (figure 4). L&#39;intensité de fluorescence de la GFP-dgnFS était dans tous les cas à 90% (données non présentées) qui indique une efficacité de transfection élevée pour les trois groupes d&#39;âge différents. <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="Figure 1" /> Figure 1. La séquence de la GFP-dgn et GFP-dgnFS. Affichée est l&#39;extrémité 3 &#39;de la GFP (green) le site de clonage multiple du vecteur pEGFP-CL1 (gris) et la séquence dgn / dgnFS (rouge). <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="Figure 2" /> Figure 2. Cytométrie en flux de la GFP-dgn et GFP-dgnFS fibroblastes diploïdes humains. A. fibroblastes jeunes prépuce humain (HFF-2) ont été infectées avec les vecteurs lentiviraux porteurs d&#39;une protéine fluorescente verte (GFP)-dgn gène ou d&#39;un GFP-dgnFS (cadre de lecture) construire, comme il est indiqué et analysé pour fluorescence de la GFP en utilisant la cytométrie de flux (FACS Canto II, Becton Dickinson). Spécification, les cellules ont été traitées pendant 3 heures également avec inhibiteur de protéasome N-acétyl-L-leucyl-L-leucyl-L-norleucinal (LLNL). Les cellules non infectées ont été utilisées comme contrôle. Des expériences ont été réalisées en triple. B. Données sont représentatifs de trois expériences indépendantes. Ces chiffres reflètent la quantité de cellules GFP-positives. <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="Figure 3" /> Figure 3. Analyse microscopie à fluorescence de la GFP-dgn et GFP-dgnFS HFF-2 cellules. Jeune HFF-2 ont été traités comme dans la figure 2. À 9 jours après l&#39;infection, les cellules sont visualized par fluorescence et par microscopie à contraste de phase. <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="Figure 4" /> Figure 4. Les changements dans l&#39;activité du protéasome dans le vieillissement cutané humain. Les fibroblastes de prépuce humain de neuf différents bailleurs de fonds dans les groupes d&#39;âge indiquées ont été peu développées et infectées avec des constructions lentiviraux codant pour la GFP-dgn. À 9 jours après l&#39;infection, les cellules ont été analysées par cytométrie en flux. Les données ont été obtenues sur trois échantillons par groupe d&#39;âge en doublons (± SE). <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="Figure 5" /> Figure 5. Approche expérimentale. Custom-oligo nucléotides pour dgn et dgnFS sont clonées dans le vecteur pEGFP-C1 et les virus sont produits pour chaque construction en utilisant des cellules HEK 293FT. Le titre du virus est déterminé. Les cellules sont transduites avec le virus et élargi. Après le traitement favorable les cellules sont analyséespour le signal de fluorescence par cytométrie de flux. <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="Figure 6" /> Figure 6. Carte de Plenti GFP-dgn. La carte du pLenti6/V5-DEST vecteur comprenant la séquence GFP-dgn est affiché. Abréviations: pCMV (promoteur CMV), GFP-dgn (séquence de la GFP-dgn), P SV40 (SV40 promoteur précoce), EM7 (EM7 promoteur), blasticidine (gène de résistance à blasticidine), ΔU3 / 3&#39;LTR (3&#39;LTR avec supprimé région U3), SV40 pA (signal de polyadénylation de SV40), ampicilline (gène de résistance à l&#39;ampicilline), pUC ori (origine pUC), PRSV / LTR 5 &#39;(RSV / 5&#39;LTR promoteur hybride), Ψ (VIH-1 signal d&#39;encapsidation Ψ ), RRE (VIH-1 élément de réponse Rev). <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="Figure 7" /> Figure 7. Plaque de titrage U2-OS. U2-OS ont été ensemencées sur une plaque à 6 puits et transfectées avec différentes concentrations de virus (dilution de 1/100 et 1/10, 1, 5 et 10 pi de coopérationncentrated surnageant viral). Un puits a été utilisé comme contrôle non transfectées (NT). Après 6-7 jours, les cellules ont été colorées au cristal violet et séché à l&#39;air.

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Aucun conflit d&#39;intérêt déclaré.

La première publication en utilisant la protéine fluorescente verte (GFP) comme substrat reporter pour l&#39;activité ubiquitine-protéasome a été publié en 2000 12. Depuis lors, la GFP est devenu un outil commun de visualiser les activités cellulaires, en particulier le processus ubiquitine-protéasome. Pour surveiller l&#39;activité ubiquitine-protéasome in vivo d&#39;un modèle de souris transgénique avec un rapporteur GFP basée a été mis en place 13. Autres recherches<...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> pEGFP-C1 Vecteur </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 Directional TOPO Cloning Kit </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine 2000 réactif </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> Filtre PVDF (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polyéthylène glycol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Phosphate de Dulbecco tamponnée Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> hexadiméthrine bromure de </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidine </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Cristal violet </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS tubes </td><td> BD Biosciences </td><td></td></tr><tr><td> La streptomycine Pénicilline (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutamine 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Sérum foetal bovin (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM non-acides aminés essentiels (AANE) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> Pyruvate de sodium 100 mM MEM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-glucose (45%) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Généticine </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> Trypsine-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

Protéasome est la principale organite intracellulaire impliquée dans la dégradation protéolytique de la anormaux, mal repliées, les protéines endommagées ou oxydées 1, 2. Maintien de l&#39;activité du protéasome est impliqué dans de nombreux processus cellulaires clés, comme la réponse au stress cellulaire 3, la régulation du cycle cellulaire et de la différenciation cellulaire 4 ou en réponse du système immunitaire 5. Le dysfonctionnement du système ubiquitine-protéasome a été associée à l&#39;apparition de tumeurs et de maladies neurodégénératives 4, 6. En outre, une diminution de l&#39;activité du protéasome a été trouvé comme une caractéristique de la sénescence cellulaire et organismique vieillissement 7, 8, 9, 10. Ici, nous présentons une méthode pour mesurer l&#39;activité ubiquitine-protéasome dans les cellules vivantes en utilisant une protéine de fusion GFP-dgn. Pour être en mesure de surveiller l&#39;activité ubiquitine-protéasome dans les cellules vivantes primaires, construit d&#39;ADN complémentaire codant pour une protéine fluorescente verte (GFP)-dgn protéine de fusion (GFP-dgn, instable) et une variante portant une mutation du cadre de lecture (GFP-dgnFS, stable 11) sont insérés dans des vecteurs d&#39;expression lentiviral. Nous préférons cette technique par rapport aux techniques de transfection traditionnels, car il garantit une efficacité de transfection très élevée indépendamment du type de cellule ou de l&#39;âge du donateur. La différence entre la fluorescence émise par la GFP-dgnFS (stable) et la protéine déstabilisé (GFP-dgn) en l&#39;absence ou en présence de l&#39;inhibiteur de protéasome peuvent être utilisées pour estimer l&#39;activité ubiquitine-protéasome dans chaque souche cellulaire particulier. Ces différences peuvent être surveillés par microscopie à épifluorescence ou peut être mesurée par cytométrie en flux.

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

Procédé pour surveiller l&#39;activité ubiquitine-protéasome dans les cellules vivantes est décrit. Un degron-GFP déstabilisée-(GFP-dgn) et une protéine de fusion GFP-stable dgnFS sont générés et transduites dans la cellule en utilisant un vecteur d&#39;expression lentiviral. Cette technique permet de générer une lignée stable de cellules exprimant GFP-dgn/GFP-dgnFS dans lequel l&#39;activité ubiquitine-protéasome peut être facilement évaluée en utilisant épifluorescence ou la cytométrie en flux.

Suivi de l&#39;activité ubiquitine-protéasome dans les cellules vivantes aide d&#39;un Degron (dgn)-déstabilisé Green Fluorescent Protein Reporter Protéines (GFP) à base de

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 vues.  Institute for Biomedical Aging Research. Procédé pour surveiller l'activité ubiquitine-protéasome dans les cellules vivantes est décrit. Un degron-GFP déstabilisée-(GFP-dgn) et une protéine de fusion GFP-stable dgnFS sont générés et transduites dans la cellule en utilisant un vecteur d'expression lentiviral. Cette technique permet de générer une lignée stable de cellules exprimant GFP-dgn/GFP-dgnFS dans lequel l'activité ubiquitine-protéasome peut être facilement évaluée en utilisa...

Vidéo: 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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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

Recherche

Biologie

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.

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

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

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)

Bimoléculaire complémentation de fluorescence (BiFC) Dosage pour les interactions protéine-protéine dans les cellules d&#39;oignon Utilisation du pistolet à gènes 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 essais (FRAP et FLIP) pour mesurer la dynamique des protéines de la chromatine dans Vivre cellules souches embryonnaires

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.

Les analyses couplées pour la surveillance protéines repliement<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.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

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.

Croissance dosage à base de Reporter-une analyse systématique de la dégradation des protéines

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).

L&#39;échelle du génome interaction protéine-protéine Le dépistage par Protein-fragment complémentation Assay (PCA) dans les cellules vivantes

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.

Green Fluorescent Protein base Expression dépistage des protéines membranaires dans<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 protocole pour l&#39;évaluation fonctionnelle de Whole-Protein Saturation mutagenèse Bibliothèques Utilisant à haut débit de séquençage

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