Biomedical Engineering Reference
In-Depth Information
Table3.9.
Amount of CDAP and TEA for activation of 10 gwet Sepharose
4B
µ
Degree of
Capacity (
Moles
mg CDAP
ml TEA
activation
act. groups per gram)
Low
5
15
0.2
Medium
15
75
0.6
High
30
150
1.2
3.6.1.1 Determination of the Degree of Activation
A 0mg N,N
-dimethylbarbituric acid dissolved in 1.8 ml rea-
Solutions/Reagents
gent-grade pyridine and made with ddH
2
Oupto2.0ml (some
days stable at −20
◦
C)
We i g h i n 5 mg of sucked-off activated gel into a test tube and add
200
µ
l Soln. A. Close the tube and mix for 15 min at RT. Add 2.80 ml
ddH
2
O and allow to settle or centrifuge. Read the supernatant at
588 nm in a photometer. If necessary, dilute with water.
Molar absorptivity of the reactive cyanate groups
ε
=
1 cm
µ
Mol
−1
cm
−1
.
Degree of activation is calculated by the following equation.
137 ml
O.D.
588
·
1000
=
a
0.137
·
3
·
mg weighed portion
References
Kohn J, Wilchek M (1983) FEBS Lett 154:209
Kohn J, Wilchek M (1984) Appl Biochem Biotechnol 9:285
Porath J (1974) Meth Enzymol 34:13
3.6.2 Coupling to Cyanogen Bromide-Activated Gels
Forming of covalent binding (coupling) between activated gel and
ligand needs free primary amino groups of the molecule to be
immobilized (ligand). The ligand may be bound directly to the
matrix or via an intervening molecule, the spacer.
To immobilize a protein the biologic activity of which depends
on a cofactor, this cofactor has to be present in sufficient concentra-
tion during the immobilization step. Thus, for example, the lectin
Concanavalin A is immobilized in the presence of a buffer contain-
ing 0.1 M glucose or
α
-methylmannoside, 1 mM calcium chloride
and 1 mM manganese chloride.
Important!
The active center of a protein and the binding site to
its ligate has to be protected by substrate, cofactor, ligand, or their
analogue during coupling to the chromatographic support.
