Download DBCO–Cy5 - Kerafast

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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.
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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.
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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.
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