Galactosidase, Beta Assay
Method: Essentally that of Craven et al. (1965). One unit causes the hydrolysis of one micromole of o-nitrophenyl-β-D-galactopyranoside per minute at 25°C and pH 7.5 under the specified conditions.
Prepare a one mg/ml stock solution in 0.10 M sodium/potassium phosphate buffer pH 7.0. Immediately prior to use, dilute further to 0.02 - 0.04 ΔA/min. in enzyme diluent. The protein concentration of the chromatographically purified enzyme (Code: BGC) may be determined as follows:
Note: This enzyme is not stable when diluted. All dilutions should be made as quickly as possible and used immediately.
Adjust spectrophotometer to 405 nm and 25°C.
Pipette into cuvette as follows:
Incubate in spectrophotometer at 25°C for 3 - 5 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml freshly diluted enzyme and record increase in A405 for 4 - 5 minutes. Calculate ΔA405/min from initial linear rate.