Freimoser:Protocols:rAPase
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Acid phosphatase (rAPase) assay
rAPase assay
This protocol follows the description of Huang & O'Shea 2005 (Genetics 169 (4): 1859-1871).
- take 50 μl from a yeast culture
- add 200 μl nitrophenyl-phosphate (20 mM)(in 100 mM NaAcetate, pH 4.2)
- incubate 15 min at 28°C (in strains with high rAPase activity the incubation time must be reduced)
- add 200 μl ice cold TCA (10 %)
- add 400 μl Na2CO3 (2 M)
- spin down, 2 min, max speed
- measure absorbance at 420 nm
- normalize OD420 with cell density (OD600), reaction time (15 min) and culture volume used for assay (50 μl). Miller units = (OD420x1000)/(OD600x15x50)
Solutions
- nitrophenyl-phosphate-solution:
- nitrophenyl-phosphate (MW = 371.12 g/mol)
- make 20 mM solution in NaAcetate (100 mM, pH 4.2) (0.00742 g/ml)
- 10% TCA (trichloroacetic acid)
- 2 M Na2CO3 (sodium carbonate)