Description:
pCLIPfVectorisamammalianexpressionplasmidintendedforthecloningandstableortransientexpressionofCLIP-tag®proteinfusionsinmammaliancells.ThisplasmidencodesCLIPf,aCLIP-tagprotein,whichisexpressedundercontroloftheCMVpromoter.TheexpressionvectorhasanIRES(internalribosomeentrysite)andaneomycinresistancegenedownstreamoftheCLIPffortheefficientselectionofstabletransfectants.pCLIPfVectorcontainstwomultiplecloningsitestoallowcloningofthefusionpartnerasafusiontotheN-orC-terminusoftheCLIPf.
TheCLIP-tagisanoveltoolforproteinresearch,allowingthespecific,covalentattachmentofvirtuallyanymoleculetoaproteinofinterest.TheCLIP-tagisasmallproteinbasedonhumanO6-alkylguanine-DNA-alkyltransferase(hAGT).CLIP-tagsubstratesarederivativesofbenzylcytosine(BC).Inthelabelingreaction,thesubstitutedbenzylgroupofthesubstrateiscovalentlyattachedtothereactivecysteineofCLIP-tagformingastablethioetherlink.
pCLIPfcontainsanimprovedversionofCLIP-tag,termedCLIPf.CLIPfdisplaysfasterkineticsininvitrolabelingandfast,specificandefficientlabelinginliveandfixedcellapplications,therebyrenderingitadesiredresearchtoolforanalysisofproteindynamics.
AlthoughCLIP-tagisbasedonthesameproteinasSNAP-tag®,thebenzylcytosinesubstratesformaseparateclassofsubstrates,differentfromthebenzylguaninesubstratesrecognizedbySNAP-tag.CLIP-tagandSNAP-tagcanbeusedfororthogonalsimultaneouslabeling.
Therearetwostepstousingthissystem:subcloningandexpressionoftheproteinofinterestasaCLIPffusion,andlabelingofthefusionwiththeCLIP-tagsubstrateofchoice.CloningandexpressionofCLIPffusionproteinsaredescribedinthisdocument.ThelabelingofthefusionproteinswithCLIP-tagsubstratesisdescribedintheinstructionssuppliedwiththeCLIP-tagsubstrates.
DNASUandAddgenearecentralrepositoriesforplasmidclonesandcollectionsthatmayalsobehelpful.
Notes:
Storage:pCLIPfVectorissuppliedinTEbuffer(10mMTris-HCl,pH8.0,1mMEDTA)ataconcentrationof0.5µg/µl.Plasmidsolutionscanbestoredat4°Cforuptooneweek.Forlong-termstorage-20°Cisrecommended.