MolecularFormula:2.8:1D514:C317
StorageConditions:-20C
Appearance:ClearSolution
Crystallizationofmembraneproteinsisachallengingundertakingandanumberofalternativetechniqueshavebeendevelopedsuchaslipidiccubicphaseandbicellecrystallization.Bothofthesetechniquesallowforthereconstitutionofadetergentsolubilizedmembraneproteininalipidicenvironment,andtheproteinstendtopackintotype-Icrystals.Bicellesareformedfromamixtureof1,2-dimyristoyl-
sn-glycero-3-phosphocholine(DMPC,D514)and1,2-dihexanoyl-
sn-glycero-3-phosphocholine(DHPC,D606)oramixtureofDMPCand3-((3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesulfonate(CHAPSO,C317).Bicellesarebilayerdiscswiththebilayerformedbythelipid(DMPC)andthedetergent(DHPCorCHAPSO)coveringtheedgesofthehydrophobicbilayer.
Settingupacrystallizationtrialinbicellescanbedonewithfoursimplesteps:
- Preparationofthebicellemedium
- Incorporationoftheproteinintobicelles
- Setupofcrystallizationtrials
- Monitoring,harvesting,anddatacollectionofcrystals
Tosimplifythisprocess,
Anatraceofferspremixedbicellesolutions.Thesesolutionscontain0.25mlofa30%solutionofeitherDMPC:CHAPSOorDMPC:DHPCbicellesata2.8:1ratio.Forinitialcrystallizationscreening,werecommendscreeningbetween5%-8%bicelles.Toreconstituteaproteinintobicelles,proteinismixedwiththebicellesolutionina4:1ratio.Forexample,tocreate100 µlofa5%protein-bicellemixture,the30% stockbicellesolutionisfirstdilutedwithH
2Oto25%(16.7µl30%bicelles+3.3µlH
2O=20 µl25%bicelles),and then addedto80 µlofconcentratedproteinsolution.The reconstitutedproteininbicellescanthenbeusedtosetupcrystallizationexperimentsineitherhangingdrop,sittingdrop,ortheMicrolyticCrystalFormerplates.AfullprotocolforproteinreconstitutionandcrystallizationisbeavailableundertheTechnicalDocumentationtab.
Note:Bicellesareshippedatroomtemperature.Storeat-20
oCuponreceiving.
Citations:
1.)
Faham,S.andBowie,J.U.(2002) J. Mol.Biol. 316(1), 1-6.2.)Poulos,S.
etal. (2015)
MethodsinEnzymology 557, 393-416.
3.)Kimble-Hill,A.C.(2013)
Front.Biol. 8(3), 261-272.
4.)Agah,S.andFaham,S.(2012)
MethodsinMolecularBIOLOGy 914,3-16.
