66
afc
Activated resins – ready for direct ligand
attachment
TOYOPEARL AF-Tresyl-650M
activated resin is highly
reactive toward amine and thiol groups. It is provided in dry
form, ready for reaction in buffered solutions containing
protein or other ligand. Coupling is accomplished in neutral
to slightly alkaline (pH 7 - 8) solution (Figure 2).
Under such conditions, even proteins of limited stabilitymay
be successfully coupled. Coupling leads to the formation of
a highly stable secondary amine or thio-ether linkage. The
optimized tresyl-density (ca. 20 µmol/mL hydrated resin)
is sufficient to provide substantial protein binding while
avoiding excessive multi-point attachment and consequent
impairment of ligand affinity/activity. Representative data
are presented in Table II.
TOYOPEARL AF-Epoxy-650M
activated resin, also packaged
in dry form, has a high density of epoxy-functionality
(ca. 800 µmol/mL). Under appropriate reaction conditions,
this may be used for immobilization of proteins or low
molecular weight ligands. It is particularly useful when high
densities of low molecular weight ligands must be attached
(Figure 3). Glutathione and glycine have, for example, been
coupled at densities greater than 100 µmol/mL hydrated
resin.
TOYOPEARL AF-Epoxy-650M resin is a highly versatile
starting material for conversion to other chemically
active functional groups required in special applications.
This resin may be readily activated to hydrazide-bearing
materials. This is particularly useful for immobilization
of carbohydrates or glycoproteins. Using the reaction
sequences described, special ligands may be introduced
onto this dimensionally stable, macroporous support.
Affinity
chromatography
figure 2
TOYOPEARL AF-Tresyl Coupling Procedure
R = hydrophilic polymer
-O-R-O-CH
2
CH
2
OSO
2
CH
2
CF
3
+ HS-Ligand
Toyopearl AF-Tresyl
HW-
65
Stable sulfide linkage
HW-
65
-O-R-O-CH
2
CH
2-
S-Ligand + CF
3
CH
2
SO
3
H
pH: 7.5-8
25 °C
-O-R-O-CH
2
CH
2
OSO
2
CH
2
CF
3
+ H
2
N - Ligand
Toyopearl AF-Tresyl
HW-
65
-O-R-O-CH
2
CH
2
-NH-Ligand + CF
3
CH
2
SO
3
H
Stable secondary amine linkage
HW-
65
pH: 7.5-8
25 °C
COUPLING PROCEDURE
figure 3
TOYOPEARL AF-epoxy Coupling Procedure
-O-R-O-CH
2
-CH-CH
2
+ H
2
N-Ligand
Toyopearl AF-Epoxy
-O-R-O-CH
2
-CHOH-CH
2
-NH-Ligand
Stable secondary amine linkage
R = hydrophilic
HW-
65
HW-
65
O
pH: 9-11
40 °C
-O-R-O-CH
2
-CH-CH
2
+ HS-Ligand
Toyopearl AF-Epoxy
-O-R-O-CH
2
-CHOH-CH
2
-S-Ligand
Stable sulfide linkage
HW-
65
HW-
65
O
pH: 7-8
25 °C
