Find the best conjugation reagent for your experiment

Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents contain reactive ends to specific functional groups (e.g., primary amines or sulfhydryls) on proteins or other molecules.

Choosing among the available options can seem overwhelming until you consider your application needs and categorize these reagents by their general features.

We provide two options for choosing the right crosslinking reagents for your bioconjugation experiments. 

Learn more about homobifunctional and herterobifunctional crosslinkers

Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm. Generally, they must be used in one-step reaction procedures to randomly ”fix” or polymerize molecules containing like functional group. This is useful for capturing a snapshot of all protein interactions, but it cannot provide the precision needed for other types of crosslinking applications.

Heterobifunctional crosslinkers possess different reactive groups at either end. These reagents not only allow for single-step conjugation of molecules that have the respective target functional group, but they also allow for sequential (two-steps) conjugations that minimize undesirable polymerization or self-conjugation. In sequential procedures, heteobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. After removing excess unreacted crosslinker, the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs.

Table 1: Homobifunctional crosslinkers (same reactive ends)

Crosslinking target Reactive groups, features Crosslinker products
Amine-to-amine NHS esters DSG
DSS
BS3
TSAT (trifunctional)
NHS esters, PEG spacer BS(PEG)5
BS(PEG)9
NHS esters, thiol-cleavable DSP
DTSSP
NHS esters, misc-cleavable DST
BSOCOES
EGS
Sulfo-EGS
Imidoesters DMA
DMP
DMS
Imidoesters, thiol-cleavable DTBP
Other DFDNB
Sulfhydryl-to-sulfhydryl Maleimides BMOE
BMB
BMH
TMEA (trifunctional)
Maleimides, PEG spacer BM(PEG)2
BM(PEG)3
Maleimides, cleavable DTME

Table 2: Heterobifunctional crosslinkers (different reactive ends)

Crosslinking target Reactive groups, features Crosslinker products
Amine-to-sulfhydryl NHS ester / maleimide AMAS
BMPS
GMBS and Sulfo-GMBS
MBS and Sulfo-MBS
Molecular Probes™ SMCC
SMCC and Sulfo-SMCC
EMCS and Sulfo-EMCS
SMPB and Sulfo-SMPB
SMPH
LC-SMCC
Sulfo-KMUS
NHS ester / maleimide, PEG spacer SM(PEG)2
SM(PEG)4
SM(PEG)6
SM(PEG)8
SM(PEG)12
SM(PEG)24
NHS ester / pyridyldithiol, cleavable SPDP or SPDP
LC-SPDP and Sulfo-LC-SPDP
SMPT
PEG4-SPDP
PEG12-SPDP
NHS esters / haloacetyl SIA
SBAP
SIAB
Sulfo-SIAB
Amine-to-nonselective
(Photo-reactive)
NHS ester / aryl azide ANB-NOS
Sulfo-SANPAH
ATFB
NHS ester / diazirine SDA and Sulfo-SDA
LC-SDA and Sulfo-LC-SDA
NHS ester / diazirine, cleavable SDAD and Sulfo-SDAD
Carboxyl-to-amine Carbodiimide DCC
EDC or EDAC
See also: NHS and Sulfo-NHS
Sulfhydryl-to-carbohydrate Maleimide / hydrazide BMPH
EMCH
MPBH
KMUH
Pyridyldithiol / hydrazide PDPH
Hydroxyl-to-sulfhydryl Isocyanate / maleimide PMPI
Amine-to-DNA NHS ester / psoralen SPB
Milestones in protein research
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