Freimoser:Protocols:rAPase

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rAPase assay

take 50 μl from a yeast culture
add 200 μl nitrophenyl-phosphate (20 mM)
incubate 15 min at 25°C
add 200 μl ice cold TCA (10 %)
add 400 μl Na₂CO_3> (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)

nitrophenyl-phosphate-solution:
- nitrophenyl-phosphate (MW=371.12 g/mol)
- make 20 mM solution in H₂O (0.00742 g/ml)