| Contributor: |
The Laboratory of Michael Chamberlin at the University of California, Berkeley
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| This is a protocol for measuring the level of Alkaline Phosphatase activity from E. coli. Alkaline phosphatase catalyzes the cleavage of p-Nitrophenyl Phosphate to give p-Nitrophenol and Inorganic Orthophosphate |
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A. E. coli Alkaline Phosphatase Determination
1. In triplicate, add 5 to 250 μl of E. coli Alkaline Phosphatase enzyme from each sample to test tubes (see Hint #2).
2. Bring volume of sample up to 2.4 ml with 0.05 M Tris Buffer in each test tube.
3. Incubate samples for 3 min at 37°C.
B. Start and Read Reactions
1. Add 0.1 ml of 0.5% NPP to each test tube. Start timing the incubation.
2. Incubate tubes at 37°C for at least 10 min.
3. Stop the timed reaction by adding 0.5 ml of 1 M Potassium Phosphate solution.
4. Measure the absorbance of the solution at a wavelength of 410 nm in a spectrophotometer (see Hint #3).
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| 0.05 M Tris Buffer |
| 0.05 M Tris-HCl, pH 8.0
1 mM MgCl2
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| 0.5% (w/v) p-Nitrophenyl-Phosphate (0.5% NPP) |
| 5 mg/ml p-Nitrophenyl-Phosphate (CAUTION! see Hint #1)
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| 1 M Potassium Phosphate, pH 8.0 |
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Magnesium Chloride
p-Nitrophenyl-Phosphate
Tris-HCl
Potassium Phosphate
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The appropriate volume of E. coli cytoplasmic extract will have to be empirically determined as different strains of E. coli and different experimental procedures will result in different levels of Alkaline Phosphatase expression. Include blanks lacking enzyme as negative controls.
3. The p-Nitrophenolate anion has an extinction coefficient of 16,200 -M -cm in 1 M Tris at pH 8.0 at wavelength 410 nm. One unit of enzyme is the amount of enzyme required to produce 1 μmole of p-Nitrophenol per min at 37°C under standard assay conditions.
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