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)
3. incubate 15 min at 25°C
4. add 200 μl ice cold TCA (10 %)
5. add 400 μl Na₂CO_3> (2 M)
6. spin down, 2 min, max speed
7. measure absorbance at 420 nm

Solutions

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

• 10% TCA (trichloroacetic acid)

• 2 M Na₂CO_3> (sodium carbonate)