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).

  1. take 50 μl from a yeast culture
  2. add 200 μl nitrophenyl-phosphate (20 mM)(in 100 mM NaAcetate, pH 4.2)
  3. incubate 15 min at 28°C (in strains with high rAPase activity the incubation time must be reduced)
  4. add 200 μl ice cold TCA (10 %)
  5. add 400 μl Na2CO3 (2 M)
  6. spin down, 2 min, max speed
  7. measure absorbance at 420 nm
  8. 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)