Biomedical Engineering Reference
In-Depth Information
Table 3.6.
Reagents for direct immobilization of ligands
Reagent suitable
Proteins
Nucleic
-NH
2
-SH
Polysac-
at pH
for:
acids
charides
Bisoxiranes
x
x
x
x
8.5 - 12
Bromocyan
x
x
x
9 - 11
Chloroformic
x
x
x
(x)
6 - 10
acid esters
Divinylsulfone
x
x
x
x
x
8 - 10
Epichlorhydrine
x
x
x
8.5 - 12
Glutaraldehyde
x
x
6.5 - 9
Maleimide
x
x
6.5 - 7.5
N-Chlorcar-
x
x
(x)
4 - 10
bonyloxy-2,3-di-
carboximido-5-
norbornene
Periodate
x
x
x
7.5 - 9
Trichloro-s-triazin
x
x
x
x
7.5 - 9
Table 3.7.
Reagents for coupling of ligands to spacers
Reagent
Needed group on:
Spacer
Ligand
Carbodiimide
a
-NH
2
-COOH
Chloroformic acid esters
b
-COOH
-NH
2
,-OH
Maleimid-NHS derivative
-NH
2
-SH
N-Bromosuccinimide (NBS)
-COOH
-NH
2
N-Hydroxysuccinimide (NHS)
c
-COOH
-NH
2
Orthoformic acid esters
-NH
2
-COOH
a
e.g., EDAC (EDC)
b
e.g., SMC
c
e.g., ClCO-ONB
tein. The first case is demonstrated by the protocol for horseradish
peroxidase purification on ConA Sepharose (Protocol 3.6.2.4), and
the latter is illustrated by the purification of rabbit immunoglobu-
lin A on protein A Sepharose (Protocol 3.6.2.6).
Competitive desorption (elution) has the advantage of very
smooth conditions for protein structure, but the disadvantage of
difficult removing of the ligand when further biospecific interac-
tions are intended.
Denaturationofoneorbothpartnersoftheaffinitypairisdone
either by changing pH, by salts, or by chaotropic substances. This
kind of desorption is easy to do, but there is danger of irreversible
