Liposome Swelling Assay
来自 : 蚂蚁淘
LiposomeSwellingAssayREFERENCES:NikaidoandRoseenberg(1983)J.Bact153:241-252;Yoshimuraetal.(1983)J.Bact258:2308-2314.METHOD:- 6.2µmolesoflipid(ideallyeggphophatidylcholineordipalmitoylphosphorylcholine)and0.2µmoledicetylphosphatearedriedatthebottomofavacuumtubeunderastreamofnitrogen.
- SUSPendlipidfilmin0.2mlaqueoussolutionofpurifiedporinorporincontainingcellenvelopes.Itisbesttouseaconcentratedproteinsolution(<1mg/ml)sothatmostofthedetergentintheproteinsolutionwillbedilutedout.
| E.coliporins:use5-20µg/200µl |
| P.aeruginosaporins:use2-10µg/200µl |
- Sonicateinwaterbathsonicatoronhigh15-30secs.Suspensionshouldturnfromopaquetotranslucent.
- Drysuspensioninthesamevacuumtubesbywarmingthemina45ºCwaterbathwhileevacuatingusingtubeconnectedtothevacuumpumpviaaCuSO4dryingtube.Thedryingprocesstakes2-3minutes.
- StoreliposomesovernightinevacuateddessicatorcontainingCuSO4.
- Gentlyresuspendliposomesin0.4mloffollowingandleaveundisturbedatRT(23ºC)for2hthengentlyresuspendbyshakingwithhand.
| 12mMstachyose(E.coli)or17%w/vDextranT-20(P.aeruginosa) |
| 4mMsodiumNAFD(addNAOHtoNADsol.topH6.0) |
| 1mMimidazole-NAD(pHimidazoleto6.0withdryNAD |
- Filtersuspensionthrough8umMilliporemembranefiltertoremovelargeaggregates.
- Makeup5mlsofsugarsolutions:
| Ecoli:18mMsugarorPaeruginosa:40mMsugar |
| 1mMsodiumNAD |
| 1mMimidazole-NADpH6.0 |
| SugarsshouldrangeinMWfrom150-700. |
- MeasureliposomeswellingbyfollowingthechangeinOD400usingthePerkin-Elmerspectrophotometerandtheattachedchartrecorder.Therateisestimatedfromtheinitialslope.Todothis,asquicklyaspossIBLe,add10to20µloftheliposomesuspensiontoacuvettecontaining0.6mlofthesugarsolutionandturningonthechartrecorder.
- Animportantcontrolareliposomesmadewithoutproteinbutwiththesameamountofdetergentasaddedfortheprotein-containingliposomes.
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