Name: monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret
Uploaded: 2012-01-27T12:00:00
Description: Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Forfatterne er støttet af den svenske forskningsråd, det svenske grundlaget for strategisk forskning, den svenske Kræftens Bekæmpelse, den svenske barnet Kræftens Bekæmpelse, Åke WIBERGS fundament og Jeanssons fundament.

1. Introduktion sonden til celler <ol><li> Co-transfect celler med en FRET-baseret sonde og et plasmid der giver resistens. Vælg et transfektion metode, der er effektiv i din celle-type af interesse. For U2OS celler, giver standarden calciumphosphat transfektion metoder tilfredsstillende resultater 15. </li><li> Marker celler i passende antibiotika i mindst syv dage. Dette beriger mængden af ​​celler, der udtrykker sonden og begrænser mængden af ​​celler med giftige udtryk niveauer, eller udtryk...

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Overvågning FRET hele cellecyklus kræver overvejelser, der er mindre afgørende ved vurderingen af ​​kortfristede reaktioner på eksterne stimuli. Først er cellecyklus progression nemt forstyrres af stress signalering, der kræver, at fototoksicitet holdes på et minimum. For det andet kan alle journalister potentielt påvirke cellulære processer ved titrering ud kinaser, fosfataser eller interaktion domæner. Den nok mest ligefremme måde at vurdere, om de eksperimentelle betingelser er passende, er, at måle c...

<table border="1"><tbody><tr><td> Reagens </td><td> Katalognummer </td><td> Firma </td></tr><tr><td> Leibovitz L-15, ingen phenolrødt </td><td> 21083-027 </td><td> GIBCO ved Life Technologies </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> GIBCO ved Life Technologies </td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0,05% Trypsin EDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> Penicillin-Streptomycin </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO ved Life Technologies </td></tr><tr><td> Puromycin </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster resonans energi overførsel (FRET)-baseret reportere 1 tager højde for vurderingen af endogene kinase og fosfatase aktiviteter i levende celler. Sådanne sonder består typisk af varianter af den fælles fiskeripolitik og YFP, intervenerede ved en phosphorylatable sekvens og en fosfor-bindende domæne. Efter fosforylering, FRET sonden ændringer kropsbygning, hvilket resulterer i en ændring af den afstand eller orientering mellem den fælles fiskeripolitik og YFP, hvilket fører til en ændring i effektivitet (figur 1). Flere sonder er blevet offentliggjort i det seneste årti, at overvåge aktiviteten balance af flere kinaser og fosfataser, herunder indberettere af PKA 2, PKB 3, PKC 4, PKD 5, ERK 6, JNK 7, CDK 18, Aurora B 9 og Plk1 9 . I betragtning af den modulære design, er yderligere sonder sandsynligvis opstå i den nærmeste fremtid 10. Progression gennem cellecyklus er ramt af stress signaling veje 11. Især er cellecyklus reguleret forskelligt under uforstyrrede vækst i forhold til, når cellerne er ved at komme fra stress 12. Time-lapse billeder af celler gennem cellecyklus derfor kræver særlig forsigtighed. Dette bliver et problem, især ved ansættelse af ratiometrisk billedbehandling, da to billeder med en høj signal støjforhold er forpligtet til at fortolke resultaterne. Ratiometrisk FRET billeddannelse af cellecyklus afhængige ændringer i kinase og phosphatase aktiviteter har overvejende været begrænset til sub-sektioner af cellecyklus 8,9,13,14. Her diskuterer vi en metode til at overvåge FRET-baserede prober bruger ratiometrisk billeddannelse i hele den menneskelige cellecyklus. Metoden bygger på udstyr, der er til rådighed for mange forskere inden for biovidenskab og kræver ikke ekspertviden af ​​mikroskopi eller billedbehandling.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRET-baserede journalister i stigende grad anvendes til at overvåge kinase og fosfatase aktiviteter i levende celler. Her beskriver vi en metode til, hvordan du bruger FRET-baserede journalister til at vurdere cellecyklus-afhængige ændringer i mål fosforylering.

Overvågning kinase og fosfatasesæt aktiviteter gennem cellecyklus ved Ratiometrisk FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

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