Questo studio è stato finanziato da: National Research Network on Aging (NFN S93) dalla Science Foundation austriaco (FWF), Commissione europea integrata Progetti MIMAGE e PROTEOMAGE, Netherlands Genomics Initiative / Organizzazione olandese per la ricerca scientifica (NGI / NWO, 05040202 e 050 - 060-810 LPN), finanziato dall&#39;UE, Rete di eccellenza Durata (6 ° PQ 036894), e il Programma Innovation Research Oriented sulla genomica (SenterNovem, IGE01014 e IGE5007).

1. Costruzione plasmide <ol><li> Ordine personalizzato oligo-nucleotidi codifica per DGN (ACKNWFSSLSHFVIHL 11) e per dgnFS (HARTGSLACPTSSSICE) e legare esso nel pEGFP-C1 vettore per ottenere la fusione della GFP con DGN / dgnFS (Figura 1). </li><li> Amplifica la sequenza codificante per GFP-DGN e GFP-dgnFS mediante PCR secondo il protocollo della clonazione pENTR direzionale Kit TOPO e continuare con il pLenti6/V5 direzionale TOPO Cloning Kit (Figura 6). </li></ol> 2. Virus di produzione <ol><li> Il giorno prima della transfezione (giorno 1) sementi su cellule HEK 293FT in fiasche T75 un modo che sarà 90-95% confluenti il ​​giorno della trasfezione. </li><li> Il giorno della trasfezione diluire 36 ml di reagente lipofectamina 2000 in 1,5 ml di DMEM senza siero e antibiotici in un falcon 15 ml. Utilizzare un altro ml 15 falco e diluire 3 mcg pLenti6/V5 portando il DNA complementare costrutto sia per GFP<html> -DGN o GFP-dgnFS, 2,5 microgrammi pMD2.G busta codifica plasmide vettore backbone e 7,5 mg psPAX2 in 1,5 ml di DMEM senza siero e antibiotici. Dopo 5 min il DNA diluito viene combinato con il reagente diluito lipofectamina 2000. </li><li> Incubare la miscela per 20 min a temperatura ambiente per permettere i complessi DNA-lipofectamina di forma. </li><li> Rimuovere il supporto di cellule HEK 293FT, sostituirlo con attenzione da 7 ml di terreno (senza antibiotici) e aggiungere il DNA-lipofectamina miscela al pallone e la roccia avanti e indietro per la miscelazione (2 ° giorno). Incubare la beuta notte a 37 ° C in un umidificata 5% CO 2 incubatore. </li><li> Cambiare media il giorno successivo (10 ml di mezzo senza antibiotici; 3 ° giorno). </li><li> Dopo 48 ore (giorno 5) supernatante raccolto, centrifugare a 300 xg per 5 min a temperatura ambiente e filtrare il supernatante attraverso un filtro di PVDF 0,45 micron. </li></ol> Cultura medio - DMEM (HEK 293FT) tenda &quot;&gt; DMEM 10% FBS 0,1 mM MEM NEAA 6 mM L-glutammina 1 mM MEM Piruvato di sodio 1% Pen-Strep (opzionale) 500 pg / ml Geneticin (opzionale) 3. Concentrazione Virus polietilene glicole (PEG) Precipitazioni <ol><li> Aggiungere un volume di glicole polietilene soluzione (50 Polietilenglicole mM, 41 mM NaCl, autoclave, pH = 7.2; PEG) a quattro volumi di surnatante e incubare per 2 ore a 4 ° C. Mescolare accuratamente ogni 20-30 minuti per inversione. </li><li> Centrifugare a 1500 xg per 30 min a 4 ° C. Un pellet bianco deve essere visibile. </li><li> Aspirare il surnatante e centrifugare nuovamente a 1500 xg per 5 minuti a 4 ° C. Aspirare la soluzione rimanente PEG. </li><li> Risospendere il precipitato in media o tampone fosfato salino (PBS) pipettando su e giù e vigorosamente vortex per 20 a 30 secondi. Come linea guida usa 500 pl per una beuta T75 e aliquota a 100 pl. Conservare il virus a -80 ° C. </li></ol><ol><li> Seme su 5 × 10 4 U2-OS cellule in ciascun pozzetto di una piastra da 6 pozzetti il giorno prima della transfezione. </li><li> Il giorno della trasfezione rimuovere il mezzo di coltura e sostituirla con 1 ml di DMEM contenente 8 mg / ml polibrene (hexadimethrine bromuro). Diluire il virus concentrato surnatante 1:10 e 1:100 e aggiungere 1 ml ciascuna ad un pozzetto. Per concentrazioni superiori virus usare 1 ml, 5 microlitri e 10 pl di surnatante virale concentrato per restanti pozzetti. Un pozzetto viene lasciata come controllo positivo. </li><li> Il giorno seguente sostituire il mezzo da 2 ml di DMEM. </li><li> Inizia con la selezione il giorno successivo. Applicare 10 pg / ml di blasticidina ciascun pozzetto. Sostituire il mezzo antibiotico contenente ogni secondo giorno. </li><li> Circa 6-7 giorni dopo aver iniziato la selezione delle cellule untransduced sono morti. Per la colorazione lavare le cellule tre volte con PBS e coprire le cellule con cristal violetto (10 mg / l cristal violettoin 20% etanolo) e incubare per 5-10 minuti a temperatura ambiente. Aspirare cristallo viola (può essere riutilizzato un paio di volte) e sciacquare i pozzetti due volte con DDH 2 O e asciugare la piastra. </li><li> Contare le colonie e moltiplicare con 1.000 e la diluizione corrispondente (Figura 7). Questa procedura permette di calcolare le unità di trasfezione (TU) del virus / ml. </li></ol> Cultura medio - DMEM (HFF-2/U2-OS) DMEM 10% FBS 6MM L-glutammina 1% Pen-Strep 5. Trasduzione di fibroblasti diploidi umane (Questa procedura può essere utilizzata per qualsiasi tipo di cellula.) <ol><li> Seed su 5 × 10 4 fibroblasti diploidi umane (HDFS) a 6-pozzetti il giorno prima trasduzione. </li><li> Utilizzare una molteplicità di infezione di due insieme con 8 mg / ml polibrene trasduzione come enhancer in un totale di un millilitro. </li><li> Cambiare media il giorno successivo. </li><li> Quandole cellule sono a 70-80% di confluenza inizio della selezione con 10 pg / ml blasticidina. Dopo la selezione è completa (assenza di cellule non trasfettate più muoiono) l&#39;espansione delle cellule può iniziare e le cellule sono pronte per esperimenti. Selezione cronica con blasticidina permette di generare una linea cellulare stabile overexpressing GFP-DGN o GFP-dgnFS proteine ​​di fusione, pronti per misurare l&#39;attività del proteasoma. Questa procedura garantisce un elevata efficienza di trasfezione molto indipendente dal tipo di cellula. </li></ol> 6. Misura di Citometria a Flusso <ol><li> Seed out 1 × 10 5 cellule (trasporto o GFP-DGN o GFP-dgnFS) il giorno prima dell&#39;esperimento. </li><li> Trattare cellule di controllo 3 ore prima della misurazione con l&#39;inibitore del proteasoma, ad esempio 100 mg / ml LLNL. </li><li> Lavare due volte con PBS e raccogliere le cellule con 0,5 ml di tripsina. Per arrestare la tripsinizzazione utilizzare 4,5 ml di mezzo di coltura e trasferire le cellule a 15 ml di una falcon. </li><li> Spin tegli cellule a 300 xg per 5 min a temperatura ambiente. </li><li> Aspirare il terreno, risospendere le cellule in PBS e centrifugare di nuovo a 300 xg per 5 min a 37 ° C. </li><li> Aspirare il PBS, risospendere in 400 ml di FACS tampone (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2, pH = 7.4) e trasferire le cellule nei tubi FACS. </li><li> Mantenere le cellule su campioni di ghiaccio e misura mediante citometria a flusso. Le cellule non devono essere conservate per più di 30 minuti a 4 ° C. </li></ol> 7. Risultati rappresentativi La GFP-dgn proteina di fusione porta una sequenza che si rivolge al proteasoma e quindi la proteina è immediatamente degradato e corrisponde alla riduzione del segnale di fluorescenza GFP. Il frameshift mutante (GFP-dgnFS) porta una versione mutata di questa sequenza e non è degradata da proteasoma, conduce alla maggiore fluorescenza verde. Per queste ragioni, giovane HDFS previsto ad alta attività del proteasoma e trasdotte con GFP-dgn mostrano una bassa (6% cellule positive) sia nel segnale di fluorescenza misurazione del flusso e citometria a epifluorescenza (Figura 2A e B, figura 3). Lo stesso HDFS trasdotte con GFP-dgnFS visualizzare 39,7% di cellule positive. Il trattamento delle cellule con inibitore LLNL proteasoma (N-acetil-L-leucil-L-leucil-L-norleucinal) sollevato il segnale massimo del 62,9% in entrambi, GFP-DGN e GFP-dgnFS cellule (Figura 2A e B ). Per determinare la possibile perdita di attività del proteasoma in età compresa tra campioni di pelle umana, fibroblasti dermici isolati da donatori giovani, di mezza età e anziani sono stati infettati con GFP-DGN e GFP-dgnFS, come sopra descritto e coltivato al numero stesso passaggio prima dell&#39;analisi con flusso citometria. In questi esperimenti, un netto aumento del segnale GFP tra gli individui giovani (11,2 ± 0,88% GFP-positive cellule) e di mezza età donatori (20,4 ± 2,27% GFP-positive cellule, P = 0.003) è stato osservato, che indica un diminuzione Proteasome attività in campioni ottenuti da donatori di età compresa tra 7 (Figura 4). Nessun ulteriore diminuzione proteasoma è stata osservata in fibroblasti isolati da individui più vecchi (Figura 4). Intensità di fluorescenza di GFP-dgnFS era in tutti i casi ~ 90% (dati non mostrati) che indica una elevata efficienza di trasfezione per i tre gruppi di età diverse. <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="Figura 1" /> Figura 1. La sequenza GFP-DGN e GFP-dgnFS. Mostrato è il terminale 3 &#39;di GFP (verde) del sito di clonaggio multiplo del vettore pEGFP-CL1 (grigio) e la DGN / dgnFS sequenza (rosso). <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="Figura 2" /> Figura 2. Analisi di citometria a flusso di GFP-DGN e GFP-dgnFS fibroblasti diploidi umane. A. fibroblasti prepuzio giovani umani (HFF-2) sono state infettate con vettori lentivirali che trasportano una proteina fluorescente verde (GFP)-DGN o un gene GFP-dgnFS (frameshift) costrutto, come indicato e analizzato per fluorescenza GFP mediante citometria a flusso (FACS Canto II, Becton Dickinson). Dove indicato, le cellule sono state trattate per 3 ore con un inibitore del proteasoma N-acetil-L-leucil-L-leucil-L-norleucinal (LLNL). Cellule non infettate sono state usate come controllo. Esperimenti sono stati eseguiti in triplicato. B. Dati sono rappresentativi di tre esperimenti indipendenti. I numeri riflettono la quantità di cellule GFP positive. <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="Figura 3" /> Figura 3. Analisi di microscopia a fluorescenza GFP-DGN e GFP-dgnFS HFF-2 cellule. Giovane HFF-2 sono stati trattati come in Figura 2. A 9 giorni dopo l&#39;infezione, le cellule erano visualized di fluorescenza e microscopia a contrasto di fase. <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="Figura 4" /> Figura 4. Variazioni attività del proteasoma in invecchiamento della pelle umana. I fibroblasti prepuzio umano di nove donatori diversi gruppi di età indicati, sono stati minimamente ampliato e infettati con costrutti lentivirali codificanti per GFP-DGN. A 9 giorni dopo l&#39;infezione, le cellule sono state analizzate mediante citometria di flusso. I dati sono stati ottenuti su tre campioni per fascia d&#39;età in duplicato (± SE). <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="Figura 5" /> Figura 5. Approccio sperimentale. Custom-oligo nucleotidi per DGN e dgnFS sono clonato nel vettore pEGFP-C1 e virus sono prodotti per ogni costrutto utilizzando HEK cellule 293FT. Il titolo del virus è determinata. Cellule sono trasdotte con il virus e ampliato. Dopo il trattamento preferito le cellule vengono analizzateper il segnale di fluorescenza mediante citometria a flusso. <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="Figura 6" /> Figura 6. Mappa di Plenti GFP-DGN. La mappa del pLenti6/V5-DEST vettore compresa la GFP-DGN sequenza viene visualizzato. Abbreviazioni: pCMV (promotore CMV), GFP-DGN (sequenza di GFP-dgn), P SV40 (SV40 promotore precoce), EM7 (EM7 promotore), blasticidina (gene di resistenza blasticidina), ΔU3 / 3&#39;LTR (3&#39;LTR con cancellato U3 regione), SV40 pA (SV40 segnale di poliadenilazione), ampicillina (gene di resistenza alla ampicillina), pUC ori (origine pUC), PRSV / 5&#39;LTR (RSV / 5&#39;LTR promotore ibrido), Ψ (HIV-1 segnale di imballaggio Ψ ), RRE (HIV-1 risposta elemento Rev). <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="Figura 7" /> Figura 7. U2-OS piastra titolazione. U2-OS sono state seminate su una piastra da 6 pozzetti e transfettate con concentrazioni virali (diluizione di 1/100 e 1/10, 1, 5 e 10 pl di concentrated surnatante virale). Un pozzetto è stato usato come controllo non trasfettate (NT). Dopo 6-7 giorni, le cellule sono state colorate con violetto cristallo ed essiccato all&#39;aria.

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Nessun conflitto di interessi dichiarati.

La prima pubblicazione con la proteina fluorescente verde (GFP) come substrato reporter per ubiquitina-proteasoma attività è stata pubblicata nel 2000 12. Da allora, la GFP è diventata uno strumento comune per visualizzare le attività cellulari, in particolare il sistema ubiquitina-proteasoma processo. Per monitorare ubiquitina-proteasoma attività in vivo un modello di topo transgenico con GFP-reporter è stato introdotto 13. Ulteriori ricerca in vivo stabilito un altro model...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> pEGFP-C1 Vector </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR direzionale TOPO Cloning Kit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 direzionale TOPO Cloning Kit </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine 2000 Reagente </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> Polietilene glicole </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Fosfato Dulbecco Buffered Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> hexadimethrine bromuro </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidina </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Cristallo viola </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS tubi </td><td> BD Biosciences </td><td></td></tr><tr><td> Penicillina Streptomicina (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutammina 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM Aminoacidi non essenziali (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM Piruvato di sodio 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-glucosio (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> 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 è l&#39;organello intracellulare principale coinvolto nella degradazione proteolitica della anormali, proteine ​​misfolded, danneggiati o ossidati 1, 2. Manutenzione delle attività del proteasoma è stato coinvolto in molti processi cellulari chiave, come risposta allo stress cellulare 3, regolazione del ciclo cellulare e la differenziazione cellulare 4 o nella risposta del sistema immunitario 5. La disfunzione del sistema ubiquitina-proteasoma è stata correlata allo sviluppo di tumori e malattie neurodegenerative 4, 6. Inoltre, una diminuzione dell&#39;attività del proteasoma trovato è una caratteristica della senescenza cellulare e organismal invecchiamento 7, 8, 9, 10. Qui, vi presentiamo un metodo per misurare ubiquitina-proteasoma attività in cellule viventi usando un GFP-DGN proteina di fusione. Per poter controllare ubiquitina-proteasoma attività nelle cellule viventi primarie, DNA complementare costruisce codificante per una proteina fluorescente verde (GFP)-DGN proteina di fusione (GFP-DGN, instabile) e una variante portando una mutazione frameshift (GFP-dgnFS, stabile 11) sono inseriti in vettori di espressione lentivirali. Preferiamo questa tecnica rispetto alle tecniche tradizionali di transfezione in quanto garantisce una elevata efficienza di trasfezione molto indipendente dal tipo di cellula o l&#39;età del donatore. La differenza tra fluorescenza visualizzata dal GFP-dgnFS (stabile) proteine ​​e la proteina destabilizzato (GFP-dgn) in assenza o presenza di inibitori del proteasoma può essere usato per stimare ubiquitina-proteasoma attività in ciascun ceppo cella particolare. Queste differenze possono essere monitorati mediante microscopia in epifluorescenza o può essere misurata mediante citometria a flusso.

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 metodo per monitorare l&#39;attività ubiquitina-proteasoma in cellule viventi è descritto. Un degron-destabilizzato GFP-(GFP-dgn) e di una stalla GFP-dgnFS proteina di fusione vengono generati e trasdotto nella cella utilizzando un vettore lentivirale espressione. Questa tecnica permette di generare una linea cellulare stabile GFP-dgn/GFP-dgnFS esprimendo in cui ubiquitina-proteasoma attività può essere facilmente valutata usando epifluorescenza o citometria a flusso.

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine ​​Reporter

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 Views.  Institute for Biomedical Aging Research. Un metodo per monitorare l'attività ubiquitina-proteasoma in cellule viventi è descritto. Un degron-destabilizzato GFP-(GFP-dgn) e di una stalla GFP-dgnFS proteina di fusione vengono generati e trasdotto nella cella utilizzando un vettore lentivirale espressione. Questa tecnica permette di generare una linea cellulare stabile GFP-dgn/GFP-dgnFS esprimendo in cui ubiquitina-proteasoma attività può essere facilmente valutata usando epifluorescenza o citometria a flus...

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.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

Ricerca

Biologia

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.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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)

Biomolecolare fluorescenza complementazione (BiFC) Assay per interazione proteina-proteina nelle cellule Cipolla Utilizzando la Helios Gun Gene

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.

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

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.

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<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.

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

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.

Saggio di crescita a base Reporter, per l&#39;analisi sistematica di proteine ​​degradazione

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).

Genome-wide proteina-proteina interazione Screening per Protein-frammento Complementation Assay (PCA) in cellule viventi

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 Espressione Proiezione di proteine ​​di membrana 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.

Un protocollo per la valutazione funzionale del Whole-Protein Saturazione mutagenesi biblioteche Utilizzando 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 ...

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine ​​Reporter

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine Reporter