Freimoser:Protocols:rAPase: Difference between revisions
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# spin down, 2 min, max speed | # spin down, 2 min, max speed | ||
# measure absorbance at 420 nm | # measure absorbance at 420 nm | ||
# normalize OD<sub>420</sub> with cell density (OD<sub>600</sub>), reaction time (15 min) and culture volume used for assay (50 μl). | # normalize OD<sub>420</sub> with cell density (OD<sub>600</sub>), reaction time (15 min) and culture volume used for assay (50 μl). | ||
Miller units = (OD<sub>420</sub>x1000)/(OD<sub>600</sub>x15x50) | Miller units = (OD<sub>420</sub>x1000)/(OD<sub>600</sub>x15x50) | ||
Revision as of 04:06, 16 January 2007
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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)
- incubate 15 min at 25°C
- 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 H2O (0.00742 g/ml)
- 10% TCA (trichloroacetic acid)
- 2 M Na2CO3> (sodium carbonate)