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http://www.clickchemistrytools.com tel: 480 584 3340 fax: 866 717 2037 DBCO–Cy5 Product No.: A130 Product Name: DBCO-Cy5 Alternative name: ADIBO-Cy5 SO3 O3S N N Chemical Structure: 2 Et3NH H N N O SO3 O Chemical Composition: C52H56N4O8S3 (protonated) Spectral Properties: Abs/Em = 646/661 nm Extinction Coefficient: 251000 M-1cm-1 Molecular Weight: 1008.22 (protonated) Appearance: Blue solid Storage: Upon receipt store at -20°C. Product shipped at ambient temperature DBCO‐Cy5isanazide‐reactivefluorescentdyethatiswellsuitedfordetectionandlabelingofchemically, enzymatically,ormetabolicallyazide‐modifiedbiopolymersorpeptides.TheDBCOgroupreactswithanazide toproduceastabletriazole(Figure1),whichisalsoreferredtoastheCu(I)‐freeorstrain‐promotedclick reaction. HO HO OH H N N3 O HO O OH Metabolically Incorporated Azido Sugar O3S SO3 N N Extracellular O3S H N Intracellular N O O DBCO-Cy5 Conjugate SO3 O3S N N SO3 HO HO O O N H OH H N N N N O O HO OH N Sugar Labeled with Fluorecsent Dye Figure 1. Reaction scheme of DBCO and azide Live Cell Labeling 1. Growmammaliancellsinanappropriatemediumwithanazide‐derivatizedmetabolite(e.g.,ManNAz) at37°Cin5%CO2. 2. WashthecellstwotimeswithD‐PBScontaining1%FBS. 3. Preparea5mMstocksolutionofDBCO‐Cy5inawater‐misciblesolventsuchasDMSOorDMFby adding0.394mLofsolventto2mgvialor0.985mLtoa5mgvial,andvortextodissolveallsolid. 4. Labeltheazide‐modifiedcellsatroomtemperatureinthedarkfor30‐60minwith5to30μMofDBCO‐ Cy5inD‐PBScontaining1%FBS. 5. WashthecellsfourtimeswithD‐PBScontaining1%FBS. 6. Fixthecellswith4%formaldehydeinD‐PBSfor20minutesatroomtemperature. http://www.clickchemistrytools.com tel: 480 584 3340 fax: 866 717 2037 7. WashthecellswithD‐PBS. 8. Optionalstep:Counterstainthecellsfor15minutesatroomtemperaturewithHoechst33342inD‐PBS. 9. WashthecellstwotimeswithD‐PBS. 10. Imagethecells. Lysing Cells 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. DonotuseDTT,TCEP,orβ‐mercaptoethanolbecausetheywillreducetheazide. Preparelysisbuffer(100mMTrisbuffer(pH8.0)containing1%(w/v)SDS). Optionalstep:Addproteaseandphosphataseinhibitorsthelysisbuffer. Suspendcellsinthelysisbuffer(50μLlysisbufferper106cells)andheatto75°C.Ifusinga6‐wellplate youneed500μLlysisbufferper100mmdishand200μLlysisbufferperwell. SonicatethelysatebrieflytoshearDNAandreducetheviscosityofthesolution. Vortexthelysatefor5minutes. Centrifugethecelllysateat16,000gat4°Cfor10minutes. Transferthesupernatanttoacleantubeanddeterminetheproteinconcentrationifrequired.Ideally, theproteinconcentrationshouldbe1–2mg/mL. Prepare1Msolutionofiodoacetamidebyadding3mLofDMSOtoIAAlabeledvial(providedwitha lysislabelingkit). Blockcysteinethiolsinlysatebyadditionofiodoacetamidestocksolutiontoafinalconcentrationof15 mM,agitatemildlyfor30min. Preparea5mMstocksolutionofDBCO‐Cy5byadding0.394mLofDBCOto2mgvialor0.985mLto5 mgvial. LabelthecelllysatebyadditionofDBCO‐Cy5toafinalconcentrationof20µM.Protectfromlightand agitatemildlyfor30minatroomtemperature. Preparea50mMstocksolutionofstopbufferbyadding3mLofwatertoStopReagentlabeledvial (providedwithalysislabelingkit). Stopreactionbyadditionofstopbuffertoafinalconcentrationof100µM,agitatebrieflyfor20min. Load~10µgofproteinon12%Tris‐TricineSDS‐PAGEgel. ImagethegelbyfluorescencescanningwithdetectionforCy5,AF647,orDyLight647. StainthegelwithCoomassie‐stainaccordingtothemanufacture’sprotocol,andimagecells. http://www.clickchemistrytools.com tel: 480 584 3340 fax: 866 717 2037 References 1. (a) Baskin, J. M., et. al. (2007). Copper-free click chemistry for dynamic in vivo imaging. PNAS., 104(43):16793-7. (b) Ning, X., et al. Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast Huisgen cycloaddition. Anweg. Chem. Int. Ed., 47:2253-5. 2. (a) Rubino F. A., et al. (2012). Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry. J. Vis. Exp., 66:4246. (b) Yao J. Z., et al. (2012). Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition. J. Am. Chem. Soc., 134:3720−3728. 3. (a) Dieterich, D.C., et al. (2007). Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino acid tagging. Nature Protocols, 2(3): 532-40. (b) Best. M.D., et al. (2009). Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules. Biochemistry, 48(28):6571-84. (c) Ngo J. T., et al. (2012). State-selective metabolic labeling of cellular proteins. ACS Chemical Biology, 7(8):1326-30. http://www.clickchemistrytools.com tel: 480 584 3340 fax: 866 717 2037