526PR01

G-Biosciences 1-800-628-7730 1-314-991-6034 technical@GBiosciences.com

AGenoTechnology,Inc.(USA)brandname

EDC
1ethyl3(3dimethylamino) propyl carbodiimide,
hydrochloride
(Cat. # BC2550, BC251, BC255)

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www.GBiosciences.com

INTRODUCTION.................................................................................................................3
ITEM(S)SUPPLIED..............................................................................................................3
STORAGECONDITIONS......................................................................................................3
ADDITIONALMATERIAL(S)REQUIRED...............................................................................4
PROTOCOL.........................................................................................................................4
APPENDIX1:2STEPCOUPLINGWITHEDCANDNHS........................................................5
INTRODUCTION.............................................................................................................5
ADDITIONALMATERIAL(S)REQUIRED...........................................................................5
PROTOCOL.....................................................................................................................5
RELATEDPRODUCTS..........................................................................................................6

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INTRODUCTION
EDCisaheterobifunctional,watersoluble,zerolengthcarbodiimidecrosslinkerthatis
usedtocouplecarboxylgroupstoprimaryamines.EDCactivatescarboxylgroupsfirst
andformsaminereactiveOacylisoureaimtermediatethatspontaneouslyreactswith
primaryaminestoformanamidebondandisoureabyproduct.

Figure1:EDCCouplingScheme
Theunstablenatureoftheintermediateinaqueoussolutionsmakes2stepcoupling,
howeverinconjunctionwithNhydroxysuccinimide,a2stepcouplingispossible.
EDCisidealforpeptideimmobilizationandhaptencarrierproteinconjugation.
ITEM(S) SUPPLIED
Cat.#

Description

BC2550 EDC

Size
50mg

BC251

EDC

1g

BC255

EDC

5g

STORAGE CONDITIONS
EDCisshippedatambienttemperature.Uponarrival,storeat20Cinthepresenceofa
desiccant.

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ADDITIONAL MATERIAL(S) REQUIRED

CarrierProtein(2mg)

WerecommendourOneQuant BSA,HyperCarrier andkeyholelimpethemocyanin

(KLH)CarrierProteins(Cat.#786090,786092,786091respectively).

ConjugationBuffer
0.1MMES,pH4.55orGBiosciencesOptimizerBufferIV(Cat.#BKC07)

Haptenorpeptide(12mg)

Desaltingcolumn

WerecommendSpinOUT GT600,3ml(Cat.#786171)
PROTOCOL
1. EquilibratetheEDCtoroomtemperaturebeforeopening.
NOTE:EDCishighlyhygroscopic;failuretoallowtoequilibratemayleadtopoor
crosslinking.
2. Add2mgcarrierproteinto500lConjugationBuffer.
3. Dissolveupto2mghaptenorpeptidein500lConjugationBufferandaddtothe
proteinsolution.

4. ForHyperCarrier ,BSAorovalbumin,dissolve10mgEDCin1mldeionizedwater
andimmediatelyadd100lEDCsolutiontotheprotein:haptensolution.
ForKLH,dissolve10mgEDCin1mldeionizedwaterandimmediatelyadd50lEDC
solutiontotheprotein:haptensolution.Ifprecipitationoccurs,reducetheamount
ofEDCsolutionaddedfurther.
5. Incubateatroomtemperaturefor2hours.
6. Purifythecoupledproteinandhaptenusingadesaltingcolumn.Werecommend

ourSpinOUT desaltingcolumns.

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APPENDIX 1: 2STEP COUPLING WITH EDC AND NHS

Introduction
Thefollowingprotocolallowsforthesequentialcouplingoftwoproteinswithout
affectingthesecondproteinscarboxylsbyquenchingthefirstreactionwithathiol
containingcompound.
AdditionalMaterial(S)Required

ConjugationBuffer1
0.1MMES,pH4.55orGBiosciencesOptimizerBufferIV(Cat.#BKC07)

ConjugationBuffer2:1XPBS

Protein#1(1mg/ml),preparedinConjugationBuffer1

Protein#2(1mg/ml),preparedinConjugationBuffer2

NHSofSulfoNHS(Cat.#BC97)

Desaltingcolumn
WerecommendSpinOUTGT600,3ml(Cat.#786171)

2Mercaptoethanol

Hydroxylamine.HCl
Protocol
1. EquilibratetheEDCandNHStoroomtemperaturebeforeopening.
NOTE:Thesearehighlyhygroscopic;failuretoallowtoequilibratemayleadtopoor
crosslinking.
2. Prepare1mlofa1mg/mlsolutionofProtein#1inConjugationBuffer1.
3. Add0.4mgEDCand0.6mgNHSor1.1.mgsulfoNHSandreactfor15minutesat
roomtemperature.
4. Add1.2l2mercaptoethanoltoquenchtheEDC.
NOTE:Atthisstagetheproteincanbeseparatedfromexcess2mercaptoethanol
withadesaltingcolumn.
5. AddanequimolaramountofProtein#2comparedtoProtein#1andallowtoreact
atroomtemperaturefor2hours.
6. Quenchthereactionwiththeadditionofhydroxylaminetoa10mMfinal
concentration.
7. Purifythecoupledproteinsusingadesaltingcolumn.Werecommendour

SpinOUT desaltingcolumns.

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RELATED PRODUCTS
DownloadourProteinCrossLinkerHandbook.

http://info.gbiosciences.com/completeproteincrosslinkershandbook
Forotherrelatedproducts,visitourwebsiteatwww.GBiosciences.comorcontactus.
Lastsaved:12/6/2012CMH

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