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Abstract for PepTag® Assay for Non-Radioactive Detection of Protein
Kinase C or cAMP-Dependent Protein Kinase
The PepTag® Non-Radioactive Protein Kinase Assays provide a
rapid, sensitive and non-radioactive method to detect cAMP-dependent protein
kinase (PKA) and protein kinase C (PKC). The PepTag® Assays utilize
brightly colored, fluorescent peptide substrates that are highly specific for
the kinases in question. The hot pink color is imparted by the addition of a dye
molecule to the PepTag® Peptide substrate. Phosphorylation by PKC or
PKA of their specific substrate alters the peptide’s net charge from +1 to
–1. This change in the net charge of the substrate allows the phosphorylated
and nonphosphorylated versions of the substrate to be rapidly separated on an
agarose gel. The phosphorylated species migrates toward the positive electrode,
while the nonphosphorylated substrate migrates toward the negative electrode.
Using the PepTag® Assay, less than 10ng of kinase can be detected in
under 2 hours. This allows rapid screening of large numbers of samples such as
those produced from the assay of multiple column fractions.
The PepTag® Assay has several advantages over other
non-radioactive protein kinase assay systems. The success of a phosphorylation
reaction can be quickly determined after the electrophoretic separation step.
Because the phosphorylation of the colored peptides supplied with the PepTag®
Assays is used to measure kinase activity, phosphorylation of other substrates
occurring naturally in the sample does not add to the kinase activity measured.
This results in minimal or no background when assaying partially purified
samples as compared to using [γ-32P]ATP. Quantitation of the
phosphorylated peptide can be accomplished using a densitometer, a
spectrophotometer, a 96-well plate reader or a fluorometer. |
