Bio-Rad Affi-Gel 15 Gel - Manual (Page 4)

tive ester immobilized on the gel is highly selective for
primary amino groups, spurious side reactions with the
ligand (i.e., cross-linking or other modifications in free
solution) are eliminated. Free sulfhydryls are among
functional groups other than primary amines known to
compete for coupling.
Affi-Gel 10 and Affi-Gel 15 supports are well suited
for coupling low molecular weight ligands. This can be
done in aqueous solution or, when solubility of the ligand
permits, in organic solvent.
Fig. 1. Coupling reaction of Affi-Gel supports with ligand
containing free amino groups.
C-ON
O
O
O
+ R-NH 2
pH 6.5 to 8.5
Buffer
C-N-R
O
O
O
H
+ HO-N
3
physiological pH. Both Affi-Gel 10 and Affi-Gel 15 sup-
ports offer the following advantages:
• Covalent amide bonds couple the protein to the termi-
nal carboxyl of the spacer arm
• Highly stable in chaotropic agents and from pH 2-11
• Rapid, gentle coupling within 4 hours
• Easy to use
• High capacity of up to 35 mg protein per ml
Section 2
Coupling Chemistry
Ligands with free alkyl or aryl amino groups will cou-
ple spontaneously with Affi-Gel 10 or 15 supports in
aqueous or non-aqueous solution (refer to Figure 1). Upon
addition of ligand, the N-hydroxysuccinimide is dis-
placed, and a stable amide bond is formed. Since the reac-
2
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