Protocols and Applications Guide (US Letter Size) - Promega

||||||| 7Cell Signaling
Nonphosphorylated Substrate
+ Protease
R110
Fluorescent
FLU
100,000
90,000
80,000
70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
0 10 20 30 40 50 60 70 80 90 100
Percent Phosphorylated Peptide
Phosphorylated Substrate
+ Protease
R110
Nonfluorescent
Figure 7.15. Schematic and graph demonstrating that Rhodamine 110 is essentially nonfluorescent in the bisamide form and that the
presence of a phosphorylated amino acid (dark circle) blocks the removal of amino acids by the protease. The graph shows the average
FLU obtained after a 30-minute protease reagent digestion using mixtures of nonphosphorylated R1110 PKA Substrate and phosphorylated
R110 PKA Substrate as indicated (n = 6).
• plate-reading fluorometer with filters for reading R110
and AMC fluorescence
• protein tyrosine phosphatase or S/T protein
phosphatase
• okadaic acid (for PP1 and PP2A)
• calmodulin (for PP2B)
1. Dilute the phosphatase in Reaction Buffer and add to
wells.
2. Dilute the PTPase R110 Substrate and the Control AMC
Substrate in Reaction Buffer and add to wells.
3. Mix the contents of the plate for 15 seconds and
incubate at room temperature (10 minutes for PP1 and
PP2A; 30 minutes for PP2B; 60 minutes for tyrosine
PPase).
4. Add Protease Solution.
5. Mix the contents of the plate briefly and incubate at
room temperature (90 minutes for PP2A, PP2B or PP1;
30 minutes for tyrosine PPase).
6. Add Stabilizer Solution.
7. Mix the contents of the plate and read fluorescence.
Additional Resources for ProFluor® Phosphatase Assays
Technical Bulletins and Manuals
TB324 ProFluor® Ser/Thr PPase Assay Technical
Bulletin
(www.promega.com/tbs/tb324/tb324.html)
TB334 ProFluor® Tyrosine Phosphatase Assay
Technical Bulletin
(www.promega.com/tbs/tb334/tb334.html)
Protocols & Applications Guide
www.promega.com
rev. 3/09
Promega Publications
CN007 Monitor purified phosphatase activity
with a homogeneous non-radioactive
high-throughput fluorogenic assay
(www.promega.com
/cnotes/cn007/cn007_05.htm)
CN008 Assay protein tyrosine kinase and protein
tyrosine phosphatase activity in a
homogeneous, non-radioactive
high-throughput format
(www.promega.com
/cnotes/cn008/cn008_15.htm )
Citations
Brisson, M. et al. (2004) Discovery and characterization of
novel small molecule inhibitors of human Cdc25B dual
specificity phosphatase. Mol. Pharmacol. 66, 824–33.
The ProFluor® Ser/Thr PPase Assay was used to screen
small molecule inhibitors Cdc25B on a panel of S/T PPases
in order to characterize the specificity of these inhibitors
for Cdc25B.
PubMed Number: 15231869
Kupcho, K. et al. (2004) A homogeneous, nonradioactive
high-throughput fluorogenic protein phosphatase assay.
J. Biomol. Screen. 9, 223–31.
This article describes the use of the ProFluor® Phosphatase
Assays to measure the activity of protein phosphatases at
low concentrations.
PubMed Number: 15140384
B. Colorimetric Phosphatase Assays
Both the Tyrosine Phosphatase (Cat.# V2471) and the
Serine/Threonine Phosphatase (Cat.# V2460) Assay Systems
detect the release of phosphate from specific peptide
substrates by measuring the appearance of a phosphate
complex of molybdate:malachite green. For assays of crude
extracts, endogenous phosphate and other inhibitory
molecules are first removed by a simple 20-minute
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PROTOCOLS & APPLICATIONS GUIDE 7-18