Freimoser:Protocols:rAPase

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)