Freimoser:Protocols:rAPase: Difference between revisions
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==rAPase assay== | ==rAPase assay== | ||
This protocol follows the description of | This protocol follows the description of [http://www.genetics.org/cgi/content/full/169/4/1859| Huang & O'Shea 2005 (Genetics 169 (4): 1859-1871)]. | ||
# take 50 μl from a yeast culture | # take 50 μl from a yeast culture | ||
# add 200 μl nitrophenyl-phosphate (20 mM) | # add 200 μl nitrophenyl-phosphate (20 mM)(in 100 mM NaAcetate, pH 4.2) | ||
# incubate 15 min at | # incubate 15 min at 28°C (in strains with high rAPase activity the incubation time must be reduced) | ||
# add 200 μl ice cold TCA (10 %) | # add 200 μl ice cold TCA (10 %) | ||
# add 400 μl Na<sub>2</sub>CO<sub>3 | # add 400 μl Na<sub>2</sub>CO<sub>3</sub> (2 M) | ||
# 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). Miller units = (OD<sub>420</sub>x1000)/(OD<sub>600</sub>x15x50) | |||
==Solutions== | ==Solutions== | ||
* nitrophenyl-phosphate-solution: | * nitrophenyl-phosphate-solution: | ||
** nitrophenyl-phosphate (MW=371.12 g/mol) | ** nitrophenyl-phosphate (MW = 371.12 g/mol) | ||
** make 20 mM solution in | ** make 20 mM solution in NaAcetate (100 mM, pH 4.2) (0.00742 g/ml) | ||
* 10% TCA (trichloroacetic acid) | * 10% TCA (trichloroacetic acid) | ||
* 2 M Na<sub>2</sub>CO<sub>3 | * 2 M Na<sub>2</sub>CO<sub>3</sub> (sodium carbonate) | ||
Latest revision as of 05:37, 27 June 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)(in 100 mM NaAcetate, pH 4.2)
- incubate 15 min at 28°C (in strains with high rAPase activity the incubation time must be reduced)
- 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 NaAcetate (100 mM, pH 4.2) (0.00742 g/ml)
- 10% TCA (trichloroacetic acid)
- 2 M Na2CO3 (sodium carbonate)
