Immobilization of E coli Galactosidase Onto Thiol Reactive Agarose

For each of the following techniques it is necessary to have:

1. Empty PD-10 columns.

2. End-over-end mixer.

3. Vacuum pump.

2.2.1. Activity Determination

1. Activity buffer: 0.1 M potassium phosphate buffer, pH 7.5, containing 3 mM magnesium chloride.

2. o-Nitrophenyl-ß-D-galactopyranoside (ONPG, Sigma).

2.2.2. Immobilization of the Enzyme

1. Thiolsulfonate-agarose, prepared as described in Subheading 3.1.3.

2. Immobilization buffer: 0.1 M potassium phosphate buffer, pH 7.0.

3. 0.1 M potassium phosphate buffer, pH 7.0, 0.5 M NaCl .

4. ß-Galactosidase (ß-D-galactoside galactohydrolase; EC 3.2.1.23) grade VIII from E.coli. (Sigma).

2.2.3. Blocking Excess of Active Groups 1. Glutathione (GSH, Sigma).

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