Description:
pSNAPfVectorisamammalianexpressionplasmidintendedforthecloningandstableortransientexpressionofSNAP-tag?proteinfusionsinmammaliancells.ThisplasmidencodesSNAPf,aSNAP-tagprotein,whichisexpressedundercontroloftheCMVpromoter.TheexpressionvectorhasanIRES(internalribosomeentrysite)andaneomycinresistancegenedownstreamoftheSNAPffortheefficientselectionofstabletransfectants.pSNAPfVectorcontainstwomultiplecloningsitestoallowcloningofthefusionpartnerasafusiontotheN-orC-terminusoftheSNAPf.
TheSNAP-tagisanoveltoolforproteinresearch,allowingthespecific,covalentattachmentofvirtuallyanymoleculetoaproteinofinterest.TheSNAP-tagisasmallproteinbasedonmammalianO6-alkylguanine-DNA-alkyltransferase(AGT).SNAP-tagsubstratesarederivativesofbenzylpurinesandbenzylpyrimidines.Inthelabelingreaction,thesubstitutedbenzylgroupofthesubstrateiscovalentlyattachedtotheSNAP-tag.
pSNAPfcontainsanimprovedversionofSNAP-tag,termedSNAPf.SNAPfdisplaysfasterkineticsininvitrolabelingandfast,specificandefficientlabelinginliveandfixedcellapplications,therebyrenderingitadesiredresearchtoolforanalysisofproteindynamics.
Therearetwostepstousingthissystem:subcloningandexpressionoftheproteinofinterestasaSNAPffusion,andlabelingofthefusionwiththeSNAP-tagsubstrateofchoice.CloningandexpressionofSNAPffusionproteinsaredescribedinthisdocument.ThelabelingofthefusionproteinswithSNAP-tagsubstratesisdescribedintheinstructionssuppliedwiththeSNAP-tagsubstrates.
DNASUandAddgenearecentralrepositoriesforplasmidclonesandcollectionsthatmayalsobehelpful.
Notes:
Storage:pSNAPfVectorissuppliedinTEbuffer(10mMTris-HCl,pH8.0,1mMEDTA)ataconcentrationof0.5μg/μl.Plasmidsolutionscanbestoredat4°Cforuptooneweek.Forlong-termstorage-20°Cisrecommended.