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Carbodiim ideCrosslinkerChem istry

Carboxyl-reactive chemical groups in biomolecular probes for labeling and crosslinking
carboxylic acids to primary amines include the carbodiimide compounds EDC and DCC.
This article describes the reaction chemistries and biology research applications of this
class of reagents.
Page Contents:
Introduction
EDC
DCC
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Chemistry of Crosslinking
(summary of all reactive groups)
Overview of Crosslinking
Introduction
Carboxyl-reactive Crosslinker Reactive Groups
Very few types of chemical groups are known to provide specific and practical
conjugation to carboxylic acids (COOH), such as occur in proteins and many other
biomolecules. Certain diazomethane and diazoacetyl reagents have been used to
derivatize small compounds for analysis by HPLC or for fluorescent labeling.
Carbonyldiimidazole (CDI) can be used in non-aqueous conditions to activate carboxylic
acids for direct conjugation to primary amines (NH2) via amide bonds.
Carbodiimide compounds provide the most popular and versatile method for labeling or
crosslinking to carboxylic acids. The most readily available and commonly used
carbodiimides are the water-soluble EDC for aqueous crosslinking and the water-
insoluble DCC for non-aqueous organic synthesis methods.
Carbodiimides, as with CDI-mediated conjugation, work by activating carboxyl groups
for direct reaction with primary amines via amide bond formation. Because no portion of
their chemical structure becomes part of the final bond between conjugated molecules,
carbodiimides are considered zero-length carboxyl-to-amine crosslinkers.
Chemical structures of carbodiimides EDC and DCC. EDC (also called EDAC)
is 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride, MW 191.70.
DCC is N',N'-dicyclohexyl carbodiimide, MW 206.32.
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Bioconjugate Techniques,
2nd Edition (2008)
Greg T. Hermanson,
Academic Press, Inc.,
1202 pages.
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EDC (also called EDAC)
1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride
DCC
Dicyclohexylcarbodiimide
EDC Carbodiimide Crosslinker
EDC Reaction Chemistry
EDC reacts with carboxylic acid groups to form an active O-acylisourea intermediate
that is easily displaced by nucleophilic attack from primary amino groups in the reaction
mixture. The primary amine forms an amide bond with the original carboxyl group, and
an EDC by-product is released as a soluble urea derivative. The O-acylisourea
intermediate is unstable in aqueous solutions; failure to react with an amine results in
hydrolysis of the intermediate, regeneration of the carboxyls, and the release of an N-
unsubstituted urea.
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EDC carbodiimide crosslinker
Amine-Biotin Reagents
for labeling carboxyls with EDC
EDC (carbodiimide) crosslinking reaction scheme. Carboxyl-to-amine crosslinking with the popular
carbodiimide, EDC. Molecules (1) and (2) can be peptides, proteins or any chemicals that have respective
carboxylate and primary amine groups. When they are peptides or proteins, these molecules are tens-to-
thousands of times larger than the crosslinker and conjugation arms diagrammed in the reaction.
EDC crosslinking is most efficient in acidic (pH 4.5) conditions and must be performed in
buffers devoid of extraneous carboxyls and amines. MES buffer (4-
morpholinoethanesulfonic acid) is a suitable carbodiimide reaction buffer. Phosphate
buffers and neutral pH (up to 7.2) conditions are compatible with the reaction chemistry,
albeit with lower efficiency; increasing the amount of EDC in a reaction solution can
compensate for the reduced efficiency.
N-hydroxysuccinimide (NHS) or its water-soluble analog (Sulfo-NHS) is often included in
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MES Buffered Saline
0.1 M MES, 0.9% NaCl, pH 4.7
NHS and Sulfo-NHS
N-hydroxysuccinimide
Instructions | MSDS | CofA
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y y ( ) g ( )
EDC coupling protocols to improve efficiency or create dry-stable (amine-reactive)
intermediates. EDC couples NHS to carboxyls, forming an NHS ester that is
considerably more stable than the O-acylisourea intermediate while allowing for efficient
conjugation to primary amines at physiologic pH.
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NHS Ester Crosslinker Chemistry
Sulfo-NHS plus EDC (carbodiimide) crosslinking reaction scheme. Carboxyl-to-amine crosslinking using
the carbodiimide EDC and sulfo-NHS. Addition of NHS or Sulfo-NHS to EDC reactions (bottom-most pathway)
increases efficiency and enables molecule (1) to be activated for storage and later use.
EDC is also capable of activating phosphate groups in the presence of imidazole for
conjugation to primary amines. The method is sometimes used to modify, label,
crosslink or immobilize oligonucleotides through their 5' phosphate groups.
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Tech Tip #30: Modify and label
oligonucleotide 5' phosphate
groups
Applications for EDC Crosslinking
The ability to crosslink primary amines to carboxylic acid groups using EDC is a
powerful and versatile tool for crosslinking peptides and proteins, preparing
biomolecular probes, and immobilizing macromolecules for use in numerous protein and
cell biology detection and analysis methods.
Of course, peptides and proteins contain both primary amines and carboxylic acids (N-
and C-termini, respectively, as well as in the side-chain of certain amino acids). Thus,
EDC enables peptides and proteins to be easily conjugated to one another or to any
compounds or solid surfaces that bear either carboxyl or amino groups.
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Crosslinking Applications
Immunogen Preparation
Covalent Immobilization Methods
for Affinity Purification
1. Peptide conjugation to carrier proteins
Because peptides and proteins contain both carboxylates and amines, EDC-mediated
crosslinking usually causes random polymerization of polypeptides. This outcome is
desirable when preparing immunogens for use in antibody production because it allows
peptide antigens to be polymerized and conjugated at high densities onto immunogenic
carrier proteins, such as KLH or BSA.
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KLH and EDC Conjugation Kit
BSA and EDC Conjugation Kit
2. Label carboxyl groups with amine compounds
EDC provides the only method for labeling or crosslinking to carboxyl groups of peptides
or proteins (i.e., the C-terminus and side chains of glutamic acid and aspartic acid). To
accomplish this without also reacting to a significant number of primary amines on the
peptide or protein, one must supply a large molar excess of the desired amine-
containing molecule. For example, to biotinylate only the C-terminus of a peptide, one
would combine the peptide with something like a 100-fold molar excess of an amine-
containing biotin compound before adding the appropriate amount of EDC. Most of the
amines encountered by EDC-activated carboxylic acid groups of the peptide would be
those of the biotin compound, so peptide-to-biotin conjugation would predominate over
peptide-to-peptide conjugation.
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EDC
Amine-Biotin Compounds
(for carboxyl biotinylation)
Amine-PEG Compounds
(for carboxyl pegylation)
3. Immobilize peptides for affinity purification
Proteins are typically immobilized or crosslinked via primary amines or sulfhydryl groups
rather than through carboxylates. By contrast, peptides (and other small carboxyl-
containing molecules) are often immobilized using EDC to polymerize and conjugate
them to an amine-derivatized surface material or solid support such as agarose resin.
For example, Thermo Scientific CarboxyLink Coupling Resin is 4% beaded agarose that
has been modified with diaminodipropylamine (DADPA); it provides the necessary
amines for conjugation of peptide carboxylates at the end of a long spacer arm.
Peptides are frequently immobilized to agarose beads by this method for antigen-
specific affinity purification of antibodies following immunization of animals with peptide-
KLH conjugates.
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Covalent Immobilization Methods
for Affinity Ligands
View products...
CarboxyLink Coupling Resins and
Peptide Immobilization Kit
4. Attach peptides to surface materials
Besides agarose beads, many other solid materials are used as platforms to
immobilizing molecules for assay methods. Primary amines and carboxylic acids are the
most common foundational surface-derivatives for solid particles such as magnetic
beads or glass slides. Aminosilane compounds such as 3-aminopropyl-triethoxysilane
(APTS or APS) are popular reagents for coating glass (borosilicate) surfaces with
primary amines. EDC is then one important means for covalently attaching peptides or
other compounds to the surface material.
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Aminopropyltriethoxysilane
(APTS) aminosilane reagent
Carboxy-PEG-Amine Compounds
DCC Carbodiimide Crosslinker
DCC Reaction Chemistry and Applications
DCC (dicyclohexyl carbodiimide) crosslinks carboxylic acids to primary amines in the
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DCC (dicyclohexyl carbodiimide)
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same manner as EDC (see reaction schemes above). However, because DCC is not
aqueous-soluble, it is primarily used in manufacturing and organic synthesis applications
rather than in the typical protein research biology lab. For example, most commercially
available, ready-to-use NHS-ester crosslinkers and labeling reagents are manufactured
using DCC. Because water is excluded, the resulting NHS ester can be prepared and
stabilized as a dried powder without appreciable hydrolysis. DCC is also commonly
used in commercial peptide synthesis operations.
(dicyclohexyl carbodiimide)
Learn more...
NHS Ester Crosslinker Chemistry
Peptide Synthesis
Written and/or reviewed by Douglas Hayworth, Ph.D.
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