INSTRUCTIONS
EDC
MAN0017125
Rev. A.0
Pub. Part No. 2160475.7
22980 22981 77149
Number
Description
22980
EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), 5g
22981
EDC, 25g
77149
EDC, 10mg
N
Molecular Weight: 191.7
CAS # 25952-53-8
NH
Cl
C
N
Storage: Upon receipt store desiccated at -20°C. Products are shipped with an ice pack.
For Research Use Only. Not for use in diagnostic procedures.
Introduction
Thermo Scientific™ EDC is a carboxyl and amine-reactive zero-length crosslinker. EDC reacts with a carboxyl group first
and forms an amine-reactive O-acylisourea intermediate that quickly reacts with an amino group to form an amide bond and
release of an isourea by-product (see the Additional Information Section). The intermediate is unstable in aqueous solutions,
and therefore, two-step conjugation procedures rely on N-hydroxysuccinimide for stabilization.1,2 Failure to react with an
amine will result in hydrolysis of the intermediate, regeneration of the carboxyl and release of an N-substituted urea. A side
reaction is the formation of an N-acylurea, which is usually restricted to carboxyls located in hydrophobic regions of
proteins.1,3
Procedure for Using EDC for Coupling Haptens to a Carrier Protein
Materials Required
• Carrier protein: 2mg bovine serum albumin (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH)
• Conjugation Buffer: 0.1M MES (2-[N-morpholino]ethane sulfonic acid), pH 4.5-5 (Product No. 28390)
•
•
•
EDC: 10mg
Hapten: 1-2mg
Thermo Scientific™ Zeba™ Spin Desalting Column (Product No. 89891) or other gel filtration column with a
5-6K molecular-weight cutoff
Procedure
1. Equilibrate EDC to room temperature.
2.
Add 2mg of lyophilized BSA, OVA or KLH to 200µL Conjugation Buffer. If using Thermo Scientific™ Imject™
Carrier Proteins, reconstitute using ultrapure water.
3.
Dissolve up to 2mg of the peptide or hapten in 500µL of Conjugation Buffer and add it to the 200µL carrier protein
solution.
4.
For BSA or OVA conjugation, dissolve 10mg of EDC in 1mL of ultrapure water and immediately add 100µL of this
solution to the carrier-peptide solution. For KLH conjugation, dissolve 10mg of EDC in 1mL of ultrapure water and
immediately add 50µL of this solution to the carrier-peptide solution. Further reduce the amount of EDC if precipitation
occurs.
React for 2 hours at room temperature.
5.
6.
Purify the conjugate using a desalting column. If storing the immunogen for more than a few days, sterile filter the
conjugate and store in a sterile container at 4°C or -20°C.
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
(815) 968-0747
(815) 968-7316 fax
Thermofisher.com
Procedure for Two-step Coupling of Proteins Using EDC and NHS or Sulfo-NHS
The following protocol, adapted from a procedure described by Grabarek and Gergely, allows sequential coupling of two
proteins without affecting the second protein’s carboxyls by exposing them to EDC. This procedure requires quenching the
first reaction with a thiol-containing compound.
The activation reaction with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2; however, the reaction of NHS-activated or
Sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8. For best results, perform the first reaction in
MES buffer (or other non-amine, non-carboxylate buffer) at pH 5-6, then raise the pH to 7.2-7.5 with phosphate buffer (or
other non-amine buffer) immediately before reaction to the amine-containing molecule. For quenching the first reaction, use
2-mercaptoethanol, or the excess reagent can be simply removed (as well as the reaction pH adjusted) by buffer-exchange
with a desalting column.
Materials Required
•
•
•
•
•
•
•
•
Activation Buffer: 0.1M MES, 0.5M NaCl, pH 6.0
Coupling Buffer: Phosphate-buffered saline (PBS), 100mM sodium phosphate, 150mM NaCl; pH 7.2 (Product No.
28372)
Protein # 1: Prepared in Activation Buffer at 1mg/mL
Protein # 2: Prepared in Coupling Buffer
NHS or Sulfo-NHS (Product No. 24500 and 24510, respectively)
2-Mercaptoethanol (Product No. 35600)
(Optional) Zeba Spin Desalting Column (Product No. 89891) or other gel filtration column
Hydroxylamine•HCl (Product No. 26103)
Procedure
1. Equilibrate EDC and NHS to room temperature before opening bottles.
2.
Add 0.4mg EDC (~2mM) and 0.6mg of NHS or 1.1mg of sulfo-NHS (~5mM) to 1mL of protein #1 solution and react
for 15 minutes at room temperature.
3.
Add 1.4µL of 2-mercaptoethanol (final concentration of 20mM) to quench the EDC.
4.
Optional: Separate the protein from excess reducing agent and inactivated crosslinker using a desalting column that has
been equilibrated with Coupling Buffer (PBS).
5.
Add protein #2 to the activated protein at an equal molar ratio with protein #1. Allow the proteins to react for 2 hours at
room temperature.
6.
To quench the reaction, add hydroxylamine to a final concentration of 10mM. This method hydrolyzes nonreacted NHS
present on protein #1 and results in hydroxamate. Other quenching methods involve adding 20-50mM Tris, lysine,
glycine or ethanolamine; however, these primary amine-containing compounds modify carboxyls on protein #1.
7.
Remove excess quenching reagent using a desalting column.
Additional Information
EDC reacts with a carboxyl group first and forms an amine-reactive O-acylisourea intermediate that quickly reacts with an
amino group to form an amide bond and release of an isourea by-product (Figure 1).
Primary amine
molecule
NH
Cl
NH
H2N
O
1
OH
+
O
N
O
2
N
H
1
1
C
Carboxylate
molecule
2
N
N
EDC
O
NH
o-Acylisourea
reactive ester
Amide bond
H2N
O
+
H2N
Urea
Figure 1. One-step EDC reaction with carboxyl and amine-containing molecules.
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
2
(815) 968-0747
(815) 968-7316 fax
Thermofisher.com
Information from Our Website
•
Tech Tip #15: Biotinylate carboxyl groups with EDC and Biotin Hydrazide
•
Tech Tip #5: Attach an antibody onto glass, silica or quartz surface
•
Tech Tip #18: Block amino groups to prevent polymer formation in peptide-carrier protein conjugations
•
Tech Tip #30: Modify and label oligonucleotide 5´ phosphate groups
•
Tech Tip #3: Determine reactivity of NHS-ester biotinylation and crosslinking reagents
•
Tech Tip #46: Preferentially biotinylate N-terminal alpha-amino groups in peptides
Related Thermo Scientific Products
24500
NHS (N-hydroxysuccinimide), 25g
24510
Sulfo-NHS (N-hydroxysulfosuccinimide), 500mg
24525
Sulfo-NHS, 5g
24520
Sulfo-NHS, No-Weigh™ Format, 8 × 2mg microtubes
26166
Bioconjugate Techniques (Book)
89889
Zeba Spin Desalting Columns, 2mL, 5/pkg
89891
Zeba Spin Desalting Columns, 5mL, 5/pkg
89893
Zeba Spin Desalting Columns, 10mL, 5/pkg
22360
SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), 50mg
22322
Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), 50mg
22622
Sulfo-SMCC, No-Weigh Format, 8 × 2mg microtubes
21555
DSS (disuccinimidyl suberate), 1g
21655
DSS, 50mg
21658
DSS, No-Weigh Format, 8 × 2mg microtubes
21580
BS 3 (bis[sulfosuccinimidyl] suberate), 50mg
21585
BS 3, No-Weigh Format, 8 × 2mg microtubes
General References
Grabarek, Z. and Gergely, J. (1990). Zero-length crosslinking procedure with the use of active esters. Anal Biochem 185:131-5.
Staros, J.V., et al. (1986). Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions.
Anal Biochem 156:220-2.
Timkovich, R. (1977). Detection of the stable addition of carbodiimide to proteins. Anal Biochem 79:135-43.
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Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
3
(815) 968-0747
(815) 968-7316 fax
Thermofisher.com
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