Immobilization and Interaction Strategies in DFS of Biomolecular Partners - Dynamic Force Spectroscopy and Biomolecular Recognition - page 139

(a)

O

O

O

Tether or
cross linker

Protein or
ligand

Tether or
cross linker

+H 2 N

ON

Protein or
ligand

H

O

NHS ester

(b)

O O

Protein or
ligand

Tether or
cross linker

Tether or
cross linker

+HS

N

N

Protein or
ligand

S

O O

maleimide

(c)

EDCI

H

N N

N N

N

C

HO

N

Protein or
ligand

O

Protein or
ligand

O

O

O

NHS

HO N

O

O

Tether or
cross linker

O

NH 2

Tether or
cross linker

Protein or
ligand

H

NO

Protein or
ligand

O

O

FIGURE 4.2 Typical coupling schemes to (a) amine, (b) thiol, and (c) carboxyl functional
groups.

pH 7.2-7.5 (amine containing buffers like Tris should be avoided because of reactiv-
ity with NHS group). In organic solvents, few volume percent of organic base pyri-
dine can be added to achieve efficient coupling. If hydrolysis of NHS esters presents
a problem, then amines can be coupled to aldehydes by forming and subsequently
reducing the Schiff base as shown in Figure 4.5. However, coupling of amines to
aldehydes is slower than coupling to NHS esters (Bonanni et al. 2005).
Coupling to thiols is often achieved using reagents containing maleimide groups
as shown in Figure 4.2b, resulting in stable thioether bond (Bartels et al. 2003;
Hukkanen et al. 2005; Ray & Akhremitchev, 2005; Ray et al. 2006; Kim et al. 2009).