Diese Studie wurde gefördert durch: National Research Network on Aging (NFN S93) von der österreichischen Science Foundation (FWF), Europäische Kommission Integrierte Projekte MiMAGE und PROTEOMAGE, Niederlande Genomics Initiative / Niederländische Organisation für wissenschaftliche Forschung (NGI / NWO, 05040202 und 050 - 060-810 NHG), das EU Network of Excellence Lebensdauer (FP6 036.894) finanziert und Innovation Oriented Research Program am Genomics (SenterNovem; IGE01014 und IGE5007).

Ein. Plasmidkonstruktion <ol><li> Um individuelle Oligonucleotide Codierung für dgn (ACKNWFSSLSHFVIHL 11) und zum dgnFS (HARTGSLACPTSSSICE) und ligieren es in den pEGFP-C1-Vektor, um die Fusion des GFP mit dgn ​​/ dgnFS (Abbildung 1) zu erhalten. </li><li> Amplify die kodierende Sequenz für GFP-DGN-und GFP-dgnFS mittels PCR nach dem Protokoll des pENTR Directional TOPO Cloning Kit und fahren mit dem pLenti6/V5 Directional TOPO Cloning Kit (Abbildung 6). </li></ol> 2. Virusproduktion <ol><li> Am Tag vor der Transfektion (Tag 1) Samen aus HEK 293FT Zellen in einem T75-Flaschen, so daß sie 90-95% konfluent auf dem Tag der Transfektion sein wird. </li><li> Am Tag der Transfektion zu verdünnen 36 ul Lipofectamin 2000 Reagent in 1,5 ml DMEM ohne Serum und Antibiotika in einem 15 ml Falcon. Verwenden Sie einen anderen 15 ml Falcon und verdünnen 3 ug pLenti6/V5 Durchführung der komplementäre DNA-Konstrukt entweder für GFP<html> -DGN-oder GFP-dgnFS, 2,5 ug Umschlag kodierenden Plasmid pMD2.G und 7,5 ug Vektor-Rückgrat psPAX2 in 1,5 ml DMEM ohne Serum und Antibiotika. Nach 5 min die verdünnte DNA wird mit der verdünnten Lipofectamin 2000 Reagenz kombiniert. </li><li> Inkubieren des Gemisches für 20 min bei Raumtemperatur, um die DNA-Lipofectamin-Komplexe zu bilden ermöglichen. </li><li> Entfernen Sie das Medium HEK 293FT Zellen, ersetzen Sie es sorgfältig von 7 ml Medium (ohne Antibiotika) und fügen Sie die DNA-Lipofectamin Mischung in den Kolben und Rock hin und her für das Mischen (Tag 2). Inkubieren Sie die Kolben über Nacht bei 37 ° C in einer befeuchteten 5% CO 2-Inkubator. </li><li> Ändern Medium am nächsten Tag (10 ml Medium ohne Antibiotika-Tag 3). </li><li> Nach 48 Stunden (Tag 5) Ernte Überstand Zentrifuge bei 300 xg für 5 min bei Raumtemperatur und filtern den Überstand durch einen 0,45 um PVDF-Filter. </li></ol> Kulturmedium - DMEM (HEK 293FT) Zelt &quot;&gt; DMEM 10% FBS 0,1 mM MEM NEAA 6 mM L-Glutamin 1 mM MEM Natriumpyruvat 1% Pen-Strep (optional) 500 ug / ml Geneticin (optional) 3. Viruskonzentration durch Polyethylenglykol (PEG) Niederschlag <ol><li> Hinzufügen einem Volumen Polyethylenglykol-Lösung (50 mM Polyethylenglycol, 41 mM NaCl, Autoklaven, pH = 7,2; PEG) bis vier Volumina des Überstandes und inkubiere während 2 Stunden bei 4 ° C. Vorsichtig mischen Sie es alle 20-30 min durch Invertieren. </li><li> Abzentrifugieren bei 1500 × g für 30 min bei 4 ° C. Ein weißes Pellet sichtbar sein soll. </li><li> Überstand und zentrifugieren bei 1500 × g für 5 min bei 4 ° C. Saugen Sie den verbleibenden PEG-Lösung. </li><li> Das Pellet in Medium oder phosphatgepufferter Salzlösung (PBS) durch Auf-und Abpipettieren und kräftig Vortex für 20 bis 30 Sekunden. Als Richtwert verwenden 500 ul für eine T75-Flasche und Aliquot auf 100 ul. Bewahren Sie das Virus bei -80 ° C. </li></ol><ol><li> Saatgut aus 5 × 10 4 U2-OS-Zellen in jede Vertiefung einer Platte mit 6 Vertiefungen am Tag vor der Transfektion. </li><li> Am Tag der Transfektion entfernen Kulturmedium und ersetzen Sie sie durch 1 ml DMEM mit 8 ug / ml Polybren (Hexadimethrinbromid). Verdünnen der konzentrierten Virusüberstand 1:10 und 1:100 und fügen Sie 1 ul jeweils ein gut. Für höhere Viruskonzentrationen verwenden 1 ul, 5 ul und 10 ul konzentrierter Virusüberstand für die restlichen Brunnen. Eine Vertiefung wird als positive Kontrolle gelassen. </li><li> Am folgenden Tag ersetzt das Medium durch 2 ml DMEM. </li><li> Beginnen Sie mit der Auswahl des nächsten Tages. Übernehmen 10 ug / ml Blasticidin auf jedem gut. Ersetzen Sie das Antibiotikum enthaltenden Medium jeden zweiten Tag. </li><li> Etwa 6-7 Tage nach Beginn der Selektion werden die transduzierten Zellen tot sind. Für die Färbung Waschen Sie die Zellen dreimal mit PBS und decken die Zellen mit Kristallviolett (10 mg / l Kristallviolettin 20% Ethanol) und Inkubation für 5 bis 10 min bei Raumtemperatur. Aspirate Kristallviolett (kann ein paar Mal wiederverwendet werden) und spülen Sie die Vertiefungen zweimal mit ddH 2 O und trocknen Sie die Platte. </li><li> Zählen Sie die Kolonien und mehrfach mit 1.000 und der entsprechenden Verdünnung (Abbildung 7). Dieses Verfahren ermöglicht die Transfektion Einheiten (TU) des Virus / ml zu berechnen. </li></ol> Kulturmedium - DMEM (HFF-2/U2-OS) DMEM 10% FBS 6 mM L-Glutamin 1% Pen-Strep 5. Transduktion von humanen diploiden Fibroblasten (Diese Prozedur kann für jeden Zelltyp verwendet werden.) <ol><li> Seed von 5 × 10 4 menschliche diploide Fibroblasten (HDF) bis 6-Well-Platten am Tag vor Transduktion. </li><li> Verwenden eine Multiplizität der Infektion von zwei zusammen mit 8 ug / ml Polybren als Transduktion Enhancer in einer Gesamtmenge von einem Milliliter. </li><li> Ändern Medium am nächsten Tag. </li><li> WennDie Zellen werden bei 70-80% der Konfluenz Beginn die Auswahl mit 10 ug / ml Blasticidin. Nach der Auswahl abgeschlossen ist (nicht mehr transfizierten Zellen sterben) die Expansion der Zellen beginnen kann und die Zellen sind bereit für Experimente. Chronische Auswahl mit Blasticidin ermöglicht, eine stabile Zelllinie überexprimierenden GFP-dgn oder GFP-dgnFS Fusionsproteine ​​bereit Proteasomenaktivität messen erzeugen. Dieses Verfahren garantiert einen sehr hohen Transfektionseffizienz unabhängig vom Zelltyp. </li></ol> 6. Messung durch Durchflusszytometrie <ol><li> Seed von 1 × 10 5 Zellen (Buchwert entweder GFP-DGN-oder GFP-dgnFS) am Tag vor dem Experiment. </li><li> Saures Kontrollzellen 3 Stunden vor der Messung mit der Proteasom-Inhibitor, z. B. 100 g / ml LLnL. </li><li> Zweimal mit PBS waschen und Ernte der Zellen mit 0,5 ml Trypsin. So stoppen Sie die Trypsinierung verwenden 4,5 ml Kulturmedium und übertragen die Zellen in ein 15 ml Falcon. </li><li> Spin ter Zellen bei 300 xg für 5 Minuten bei Raumtemperatur. </li><li> Absaugen Medium, werden die Zellen in PBS und drehen sich wieder bei 300 × g für 5 min bei 37 ° C. </li><li> Absaugen PBS, in 400 ul FACS-Puffer (10 mM Hepes, 140 mM NaCl, 2,5 mM CaCl 2, pH = 7,4) resuspendiert und übertragen die Zellen in FACS-Röhrchen. </li><li> Halten Sie die Zellen auf Eis und messen Proben mittels Durchflusszytometrie. Die Zellen sollten nicht mehr als 30 min bei 4 ° C gelagert werden </li></ol> 7. Repräsentative Ergebnisse Das GFP-Fusionsprotein dgn trägt eine Sequenz, die das Proteasom und damit das Protein sofort abgebaut abzielt, sie entspricht der Abnahme der GFP-Fluoreszenz-Signal. Die Frameshift Mutante (GFP-dgnFS) eine mutierte Version dieser Sequenz und nicht abgebaut wird durch Proteasom, sie führt die höhere grünen Fluoreszenz. Aus diesen Gründen transduzierten junge HDFs mit erwarteten hohen Proteasom-Aktivität und mit GFP-dgn zeigen eine gering (6% positive Zellen) Fluoreszenzsignal sowohl Durchflusszytometrie Messung und in Epifluoreszenz (2A und B, Abbildung 3). Das gleiche HDFs transduziert mit GFP-dgnFS anzuzeigen 39,7% der positiven Zellen. Die Behandlung der Zellen mit Proteasom-Inhibitor LLnL (N-Acetyl-L-leucyl-L-leucyl-L-norleucinal) angehoben das Signal zu maximal 62,9% in beiden, GFP-dgn und GFP-dgnFS Zellen (2A und B ). Um mögliche Rückgang der Proteasom-Aktivität bei älteren menschlichen Hautproben zu bestimmen, wurden Fibroblasten von jungen, mittleren Alters und alte Spendern isoliert mit GFP-DGN-und GFP-dgnFS infiziert, wie oben beschrieben und kultiviert auf die gleiche Passage-Nummer vor der Analyse mittels Durchflusszytometrie Zytometrie. In diesen Experimenten Spender eine klare Zunahme der GFP-Signal zwischen jungen Menschen (11,2 ± 0,88% GFP-positive Zellen) und mittleren Alters (20,4 ± 2,27% GFP-positiven Zellen, P = 0,003) beobachtet wurde, was auf eine Abnahme proteasome Aktivität in Proben aus alten Spendern 7 (Abbildung 4). Keine weitere Abnahme der Proteasom-Aktivität wurde in Fibroblasten von ältesten Individuen (Abbildung 4) getrennt beobachtet. Fluoreszenzintensität für GFP-dgnFS betrug in allen Fällen ca. 90% (Daten nicht gezeigt), zeigt einen hohen Transfektionseffizienz für die drei verschiedenen Altersgruppen. <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die GFP-DGN-und GFP-dgnFS Sequenz. Angezeigt wird das 3&#39;-Ende des GFP (grün) die multiple Klonierungsstelle des pEGFP-CL1 Vektor (grau) und der DGN / dgnFS Sequenz (rot). <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Durchflusszytometrie-Analyse der GFP-DGN-und GFP-dgnFS menschliche diploide Fibroblasten. A. Young humane Vorhautfibroblasten (HFF-2) wurden mit lentivirale Vektoren, die ein grün fluoreszierendes Protein infiziert (GFP)-Gen oder ein dgn GFP-dgnFS (Frameshift) zu konstruieren, wie angegeben und analysiert GFP-Fluoreszenz unter Verwendung von Durchflusszytometrie (FACS Canto II, Becton Dickinson) analysiert. Wo angegeben, wurden die Zellen auch für 3h mit Proteasom-Inhibitor N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) behandelt. Nichtinfizierten Zellen wurden als Kontrolle verwendet. Experimente wurden in dreifacher Ausführung durchgeführt. B. Daten sind repräsentativ für drei unabhängige Experimente. Die Zahlen spiegeln die Menge an GFP-positiven Zellen. <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> . <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Fluoreszenzmikroskopie Analyse von GFP-dgn und GFP-dgnFS HFF-2-Zellen. Junge HFF-2 wurden wie in Abbildung 2 behandelt. Um 9 Tage nach der Infektion wurden die Zellen visualized durch Fluoreszenz und Phasenkontrast-Mikroskopie. <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Änderungen im Proteasom-Aktivität in menschlichen Hautalterung. Die menschlichen Vorhautfibroblasten aus neun verschiedenen Spendern in den angegebenen Altersgruppen wurden minimal erweitert und infiziert mit lentiviralen Konstrukte Codierung für GFP-dgn. Um 9 Tage nach der Infektion wurden die Zellen durchflusszytometrisch analysiert. Die Daten wurden auf drei Proben pro Altersgruppe in Duplikaten (± SE) erhalten. <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Experimentellen Ansatz. Custom-Oligonukleotide für die DGN und dgnFS in den pEGFP-C1-Vektor kloniert und Viren werden für jedes Konstrukt mit HEK 293FT Zellen produziert. Der Titer der Viren bestimmt. Zellen mit dem Virus transduziert und erweitert. Nach der bevorzugte Behandlung die Zellen werden analysiertfür Fluoreszenzsignal durch Durchflusszytometrie. <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="Abbildung 6" /> Abbildung 6. Karte von Plenti GFP-dgn. Die Karte des pLenti6/V5-DEST Vektor, der GFP-dgn Sequenz angezeigt wird. Abkürzungen: PCMV (CMV-Promotor), GFP-dgn (Sequenz von GFP-DGN), P SV40 (SV40 early Promotor), EM7 (EM7 Promotor), Blasticidin (Blasticidin Resistenzgen), ΔU3 / 3&#39;LTR (3&#39;LTR mit deletierte U3-Region), SV40 pA (SV40 Polyadenylierungssignal), Ampicillin (Ampicillin-Resistenz-Gen), pUC ori (pUC), PRSV / 5&#39;LTR (RSV / 5&#39;LTR Hybrid-Promotor), Ψ (HIV-1 Ψ Verpackungssignal ), RRE (HIV-1-Rev-Response-Element). <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="Abbildung 7" /> Abbildung 7. U2-OS Titerung Platte. U2-OS wurden auf einer 6-well-Platte ausgesät und transfiziert mit verschiedenen Virus-Konzentrationen (Verdünnung von 1/100 und 1/10, 1, 5 und 10 ul der Zusammenarbeitncentrated viralen Überstand). Ein gut als transfizierten Kontrolle (NT) eingesetzt. Nach 6-7 Tagen wurden die Zellen mit Kristallviolett angefärbt und luftgetrocknet.

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Keine Interessenskonflikte erklärt.

Die erste Veröffentlichung mit grün fluoreszierendem Protein (GFP) als Reporter Substrat für Ubiquitin-Proteasom-Aktivität wurde in 2000 12 veröffentlicht. Seitdem hat GFP zu einem gemeinsamen Instrument zur zellulären Aktivitäten, insbesondere das Ubiquitin-Proteasom-Prozess zu visualisieren. Um Ubiquitin-Proteasom-Aktivität in vivo zu überwachen ein transgenes Mausmodell mit einem GFP-basierte Reporter wurde 13 eingeführt. Zusätzliche in vivo Forschung etabliert ander...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> pEGFP-C1 Vektor </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 Reagent </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> PVDF-Filter (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polyethylenglykol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Dulbeccos Phosphat-gepufferte Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> Hexadimethrinbromid </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidin </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Kristallviolett </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS-Röhrchen </td><td> BD Biosciences </td><td></td></tr><tr><td> Penicillin Streptomycin (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-Glutamin 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Fötales Rinderserum (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM Non-Essential Amino Acids (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM Natriumpyruvat 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-Glucose (45%) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Geneticin </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> Trypsin-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

Proteasome ist der wichtigste intrazelluläre Organellen in den proteolytischen Abbau von abnormal, fehlgefalteten, beschädigte oder oxidierte Proteine ​​1, 2 beteiligt. Wartung der Proteasom-Aktivität wurde in vielen wichtigen zellulären Prozessen beteiligt, wie Zelle Stressantwort 3, Regulation des Zellzyklus und Zelldifferenzierung 4 oder Reaktion des Immunsystems 5. Die Dysfunktion des Ubiquitin-Proteasom-System hat zu der Entwicklung von Tumoren und neurodegenerativen Krankheiten 4, 6 in Verbindung gebracht. Zusätzlich wurde eine Abnahme der Proteasom-Aktivität als Funktion der zellulären Seneszenz und organismal Alterung 7, 8, 9, 10 gefunden. Hier präsentieren wir eine Methode, um Ubiquitin-Proteasom-Aktivität in lebenden Zellen mit einem GFP-dgn Fusionsprotein zu messen. Um Ubiquitin-Proteasom-Aktivität in lebenden primären Zellen zu überwachen, konstruiert komplementäre DNA, die für ein grün fluoreszierendes Protein (GFP)-dgn Fusionsprotein (GFP-dgn, instabil) und eine Variante Tragen einer Frameshift-Mutation (GFP-dgnFS, stabil 11) werden in lentiviralen Expressionsvektoren insertiert. Wir bevorzugen diese Technik gegenüber herkömmlichen Transfektionstechniken weil es eine sehr hohe Transfektionseffizienz garantiert unabhängig von dem Zelltyp oder dem Alter des Spenders. Der Unterschied zwischen der Fluoreszenz, die von GFP-dgnFS (stabilen) Protein und dem destabilisierten Protein (GFP-dgn) in Abwesenheit oder Gegenwart von Proteasom-Inhibitor angezeigt kann zum Ubiquitin-Proteasom-Aktivität in jeder bestimmten Zellstamm abzuschätzen. Diese Unterschiede können durch Epifluoreszenzmikroskopie überwacht werden können oder mittels Durchflusszytometrie gemessen werden.

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

Eine Methode, um Ubiquitin-Proteasom-Aktivität in lebenden Zellen zu überwachen beschrieben. A Degron-destabilisierten GFP-(GFP-DGN) und eine stabile GFP-dgnFS Fusionsprotein erzeugt und transduziert in die Zelle mit einem lentiviralen Expressionsvektor. Diese Technik ermöglicht eine stabile GFP-dgn/GFP-dgnFS exprimierenden Zelllinie, in dem Ubiquitin-Proteasom-Aktivität leicht beurteilt werden kann, mit Epifluoreszenz oder Durchflusszytometrie generieren.

Überwachung des Ubiquitin-Proteasom-Aktivität in lebenden Zellen unter Verwendung eines Degron (DGN)-destabilisiert Green Fluorescent Protein (GFP)-basierte Reporter Protein

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 Aufrufe.  Institute for Biomedical Aging Research. Eine Methode, um Ubiquitin-Proteasom-Aktivität in lebenden Zellen zu überwachen beschrieben. A Degron-destabilisierten GFP-(GFP-DGN) und eine stabile GFP-dgnFS Fusionsprotein erzeugt und transduziert in die Zelle mit einem lentiviralen Expressionsvektor. Diese Technik ermöglicht eine stabile GFP-dgn/GFP-dgnFS exprimierenden Zelllinie, in dem Ubiquitin-Proteasom-Aktivität leicht beurteilt werden kann, mit Epifluoreszenz oder Durchflusszytometrie generieren.

Serie: 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.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

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

Forschung

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.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

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)

Bimolekularen Fluoreszenz Komplementation (BIFC) Assay für Protein-Protein-Wechselwirkungen in Onion Zellen unter Verwendung des 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 ...

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 Assays (FRAP &amp; FLIP) zu Chromatin Protein Dynamics in Living embryonalen Stammzellen Measure

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.

Gekoppelt Assays zur Überwachung Proteinrückfaltung in<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.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

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.

Reporter-basierten Wachstumstest für die systematische Analyse von Proteinabbau

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).

Genomweite Protein-Protein-Wechselwirkungen durch Screening Protein-Fragment Komplementierungsassay (PKA) in lebenden Zellen

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-basierte Screening Expression von Membranproteinen in<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.

Ein Protokoll für Functional Assessment of Whole-Protein Sättigungsmutagenese-Bibliotheken mit Hilfe von 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 ...