Alkaline Phosphatase and Phospholipase C Assays
Categorized: Biochemical Assays
- Grow bacteria overnight in Pi sufficient and Pi deficient media.
- Read OD600 of overnight culture. One OD600 unit = 0.3 mg protein.
- Pellet 1 ml of culture in microfuge (5 min). Save supernatant.
- Add 500 µl of resulting supernatant to 500 µl of alkaline phosphatase substrate or phospholipase C substrate.
Alakaline phosphatase substrate:
- 2 mg/ml of p-nitrophenyl phosphate
- One Sigma 104 tablet (5mg) per 2.5 ml of 0.1 M Tris-HCl, pH 8.5
Phospholipase C substrate:
- 6 mg/ml nitro-phenylphosphorylcholine
- 0.25 M Tris-HCl pH 7.3
- 60% (v/v) glycerol
- Follow the A410 “absorbance change” using the Perkin-Elmer spectrophotometer and attached chart recorder. 1-2 min is enough.
- Let one sample hydrolyse the substrate to completion (i.e. no more change in absorbance) then read the A410. This is the A410 FINAL which is used to calculate the extinction coefficient.
One OD600 culture = 0.3 mg/ml proteinMW Phospholipase C substrate = 304.2; 3 mg/ml final concentration = 9.9 umoles/ml
MW Alkaline phosphate substrate = 263.1;
1 mg/ml final concentration = 3.8 umoles/ml
Extinction coefficient = umoles substrate hydrolyzed/A410 FINAL
(e.g. for APase: Ext. coef. = 3.8 umoles/A410 FINAL)
Enzyme Activity = A410 “absorbance change”/min x Extinction coefficient/mg protein = umoles substrate hydrolyzed/min./mg protein.