Description:
CLIP-Cell?TMR-StarisaphotostableredfluorescentsubstratethatcanbeusedtolabelCLIP-tag?fusionproteinsinsidelivingcells,oncellsurfacesorinvitro.Thiscell-permeablesubstrate(BC-TMR)isbasedontetramethylrhodamineandissuitableforstandardrhodaminefiltersets.Ithasanexcitationmaximumat554?nmandemissionmaximumat580nm.Thispackageincludes30?nmolofCLIP-CellTMR-Star,sufficienttomake10mlofa3μMCLIP-tagfusionproteinlabelingsolution.
TheCLIP-tagproteinlabelingsystemenablesthespecific,covalentattachmentofvirtuallyanymoleculetoaproteinofinterest.CLIP-tagisaproteintagbasedonhumanO6-alkylguanine-DNAalkyltransferase(hAGT).CLIP-tagsubstratesarederivativesofbenzylcytosine(BC).Inthelabelingreaction,thesubstitutedbenzylgroupofthesubstrateiscovalentlyattachedtothereactivecysteineofCLIP-tagformingastablethioetherbond.?AlthoughCLIP-tagisbasedonthesameproteinasSNAP-tag?,thebenzylcytosinesubstratesformaseparateclassofsubstrates,differentfromthebenzylguaninesubstratesrecognizedbySNAP-tag.CLIP-tagandSNAP-tagcanbeusedfororthogonalandcomplementarylabelingoftwoproteinssimultaneouslyinthesamecells.?
Therearetwostepstousingthissystem:subcloningandexpressionoftheproteinofinterestasaCLIP-tagfusion,andlabelingofthefusionwiththeCLIP-tagsubstrateofchoice.ExpressionofCLIP-tagfusionproteinsisdescribedinthedocumentationsuppliedwithCLIP-tagplasmids.ThelabelingofthefusionproteinswiththeCLIP-tagsubstrateisdescribedinthisdocument.
LiveCOS-7expressingpCLIP-H2BandpSNAP-ADRβ2werelabeledwithCLIP-CellTMR-Star(red)andSNAP-AlexaFluor?488(green)for60minutes.
Excitation(dottedline)andemissionspectraofCLIP-CellTMR-StarcoupledtoCLIP-taginbufferatpH7.5.
StructureofCLIP-CellTMR-Star(MW642.7g/mol)Notes:
Storage:CLIP-CellTMR-Starshouldbestoredat-20°C(longterm)orat4°Cinthedark(shortterm,lessthan4weeks).Protectthesubstratefromlightandmoisture.Withproperstorageat-20°Cthesubstrateshouldbestableforatleastthreeyearsdryor3monthsdissolvedinDMSO.