Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes

Table 1 DYRK1A kinase activity in mouse heart

	Phosphorylated peptide (pmol)
	IP		Specific result*	relative background
	αDYRK1A	αFLAG
Radiometric assay	5.63	1.20	4.43	21%
ELISA	7.88	2.24	5.63	28%

*subtraction of background (phosphorylation in αFLAG sample)
Immunoprecipitates from mouse heart lysate were split and subjected either to a radiometric or a non-radiometric kinase reaction each carried out under the same conditions (100 μM ATP, 50 μM tau_207-209, 30 min, 30°C). The amounts of phosphorylated peptide were calculated from incorporation of ³³P or determined by ELISA using a standard curve. As a background control, a parallel immunocomplex kinase assay was performed with anti-FLAG antibody instead of the anti-DYRK1A antibody. Aliquots of kinase reactions were diluted 1:10 for the ELISA. Results are means of duplicate measurements.

ISSN: 1471-2091