View - ResearchGate

260 Benčina et al.
3.1.1. Static Immobilization Method
1. Place CIM disk into CIM housing to obtain CIM disk monolithic column.
2. Connect CIM disk monolithic column to HPLC system and equilibrate it by
washing with at least 5 column volumes of water and at least 5 column volumes
of immobilization buffer (see Note 1).
3. Prepare 3 ml of a protein solution (2 g/l) by dissolving the protein in appropriate
immobilization buffer.
4. Remove CIM disk from the CIM housing and immerse it into 3 ml of the immobilization
solution (see Fig. 1A).
5. Incubate CIM disk in the immobilization solution at given temperature for a
defined period of time (see Note 1).
6. After immobilization is completed, place CIM disk again into CIM housing,
connect CIM disk monolithic column (named further “enzyme reactor”) to HPLC
system, remove the residual non-bound protein by washing the enzyme reactor
with 10 column volumes of immobilization buffer containing 0.1 M NaCl and
finally with deionized water.
7. Disconnect enzyme reactor from HPLC system, remove CIM disk from CIM
housing and store immobilized CIM disk at 4°C either in water, 20% ethanol, or
suitable buffer.
3.1.2. Dynamic Immobilization Method
1. Place CIM disk into CIM housing to obtain CIM disk monolithic column.
2. Connect a syringe filled with water to one side of the CIM disk monolithic column.
3. Equilibrate CIM disk monolithic column by pushing with a syringe at least 5
column volumes of water (∼2 ml), fill the syringe with immobilization buffer and
wash the column with at least 5 column volumes.
4. Prepare 3 ml of a protein solution (2 g/l) by dissolving the protein in appropriate
buffer and fill the syringe with it.
Fig. 1. Static (A) and dynamic (B) immobilization method.