Name: measuring peptide translocation into large unilamellar vesicles
Uploaded: 2012-01-27T13:00:00
Description: Watch this Scientific Journal Video about Measuring Peptide Translocation into Large Unilamellar Vesicles at JoVE.com

Forfatterne ønsker å takke Eleanor Fleming og Jessica Chen for nyttige diskusjoner. Finansieringen ble gitt av National Institute of Allergy og smittsomme sykdommer (NIH-NIAID) Award R15AI079685 og Research Corporation Cottrell College Science Award. Ekstra student støtte ble gitt av Howard Hughes Medical Institute og Staley fond.

1. Utarbeidelse av Stor Unilamellar Lipid Blemmer (LUVs) <ol><li> Forbered LUVs å tjene som cellemembranen etterligner for analysen 19. </li><li> Mix phosphatidylcholine (POPC, 760,10 g / mol), phosphatidylglycerol (POPG. 770,99 g / mol), 5-dimethylaminonaphthalene-1-sulfonyl phosphatidylethanolamine (DNS-PAVEN, 994,350 g / mol) (Avanti Polar Lipids) oppløst i kloroform i en 50 : 45:5 ratio. Som alle analysen krever separate utarbeidelse av både eksperimentelle og kontroll LUVs, bør to lipid kake ampull...

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Protokollen som presenteres her kan brukes til å vurdere den relative endringen i konsentrasjonen av peptider i og utenfor lipid blemmer. Disse endringene er knyttet til translokasjon evne. Denne protokollen kan brukes til å identifisere celle-gjennomtrengende peptider med potensial som narkotika levering vektorer. Som interesse i celle-gjennomtrengende peptider vokser, vil det være interessant å se hvordan metoder som direkte måler translokasjon arrangementet er utviklet og brukes i en kvantitativ måte. <p cl...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> 16:00 til 18:01 PG </td><td> Avanti Polar Lipider </td><td> 840457C </td></tr><tr><td> 01:18 Dansyl PE </td><td> Avanti Polar Lipider </td><td> 810330C </td></tr><tr><td> 16:00-18:01 PC </td><td> Avanti Polar Lipider </td><td> 850457C </td></tr><tr><td> Svin trypsin </td><td> Sigma </td><td> T-0303 </td></tr><tr><td> Bowman-Birk trypsin / chymotrypsin inhibitor </td><td> Sigma </td><td> T-9777 </td></tr><tr><td> Mini-ekstruder </td><td> Avanti Polar Lipider </td><td> 610000 </td></tr><tr><td> Ammonium molybdate (para) </td><td> Alfa Aesar </td><td> 10811 </td></tr><tr><td> L-askorbinsyre </td><td> Sigma </td><td> A1417 </td></tr><tr><td> Hydrogenperoksid, 30% løsning </td><td> Mallinckrodt kjemikalier </td><td> 5240-05 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H-3375 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S-9625 &lt;/ Td&gt; </tr><tr><td> EDTA </td><td> Sigma </td><td> E5134 </td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma </td><td> S-0751 </td></tr></tbody></table>

measuring peptide translocation into large unilamellar vesicles

Det er en aktiv interesse i peptider som lett krysser cellemembranen uten hjelp av cellemembranen reseptorer 1. Mange av disse er referert til som celle-gjennomtrengende peptider, som er ofte kjent for sitt potensial som narkotika levering vektorer 1-3. Videre er det økende interesse for antimikrobielle peptider som opererer via non-membran lytisk mekanismer 4,5, spesielt de som krysser bakteriell membraner uten å forårsake cellelyse og dreper celler ved å forstyrre med intracellulære prosesser 6,7. Faktisk har forfatterne stadig påpekt forholdet mellom celle-gjennomtrengende og antimikrobielle peptider 1,8. En fast forståelse av prosessen med membran translokasjon og forholdet mellom peptid struktur og dens evne til å translocate krever effektive, reproduserbare analyser for translokasjon. Flere grupper har foreslått metoder for å måle translokasjon inn i store unilamelLar lipid vesikler (LUVs) 9-13. LUVs tjene som nyttige modeller for bakteriell og eukaryote cellemembraner og brukes ofte i peptid fluorescerende studier 14,15. Her beskriver vi vår bruk av metoden først utviklet av Matsuzaki og medarbeidere til å vurdere antimikrobielle peptider, som for eksempel magainin og buforin II 16,17. I tillegg til å gi vår protokoll for denne metoden, vi også presentere en grei tilnærming til data analyse som kvantifiserer translokasjon evne til å bruke denne analysen. Fordelene med denne translokasjon analysen i forhold til andre er at den har potensial til å gi informasjon om graden av membran translokasjon og krever ikke tillegg av et fluorescerende etikett, noe som kan endre peptid egenskaper 18, til tryptofan som inneholder peptider. Kort fortalt er translokasjon evne til lipid vesikler målt som en funksjon av Foster Resonance Energy Transfer (FRET) mellom innfødte tryptofan rester og dansyl phosphatidylethanolamine når proteiner er forbundet med den eksterne LUV membran (figur 1). Cell-gjennomtrengende peptider er kløyvde som de støter uhemmet trypsin innkapslet med LUVs, som fører til disassociation fra LUV membran og en nedgang i FRET signal. Fallet i FRET signal observert for en translocating peptid er betydelig større enn det som ble observert for den samme peptid når LUVs inneholder både trypsin og trypsin inhibitor, eller når et peptid som ikke spontant krysse lipid membraner er eksponert mot trypsin inneholder LUVs. Denne endringen i fluorescens gir en direkte kvantifisering av peptid translokasjon over tid.

Watch this Scientific Journal Video about Measuring Peptide Translocation into Large Unilamellar Vesicles at JoVE.com

Measuring Peptide Translocation into Large Unilamellar Vesicles

Denne protokollen detaljer en metode for kvantitativ måling av peptid translokasjon inn i store unilamellar lipid blemmer. Denne metoden gir også informasjon om graden av membran translokasjon og kan brukes til å identifisere peptider som effektivt og spontant krysse lipid bilayers.

Måling Peptide translokasjon inn Large Unilamellar Blemmer

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

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