|
Download
protocol
274kb
pdf? |
|
Abstract for ProFluor® PKA Assay
The ProFluor® PKA Assay measures protein kinase A activity
using purified kinase in a multiwell plate format and involves
"add, mix, and read" steps only. The assay begins with a
standard kinase reaction performed in the provided Reaction Buffer
with a provided Bisamide Rhodamine 110 peptide substrate (PKA R110
Substrate). In this configuration the PKA R110 Substrate is
nonfluorescent. Following the kinase reaction, addition of a
Termination Buffer, which contains a Protease Reagent,
simultaneously stops the kinase reaction and removes amino acids
specifically from the nonphosphorylated PKA R110 Substrate,
producing highly fluorescent Rhodamine 110. The phosphorylated PKA
R110 Substrate, however, is resistant to digestion by the Protease
Reagent and remains nonfluorescent. Thus, the fluorescence intensity
measured in the assay is inversely correlated with kinase
activity.
|
