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Figure 1 | BMC Biochemistry

Figure 1

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

Figure 1

Direct sandwich ELISA format for the detection of the phosphorylated DYRK substrate peptide tau 207-219 . A, Scheme illustrating the principle of the assay. Bio, biotin; TMB, tetramethylbenzidine; HRP, horseradish peroxidase (coupled to streptavidin). B and C, Titration of phosphorylated and unphosphorylated tau207-219. The wells were coated with 100 ng anti tau(pT212) and loaded with dilution series of either phosphorylated or unphosphorylated tau207-219. The background signal from wells loaded only with the buffer was subtracted from all values. A representative experiment of three is shown. Panel C presents the same data as in panel B with a linear x-axis to visualize the linear range of the ELISA. The inset shows an enlargement of lower range. D, Titration of phosphorylated and non-phosphorylated tau207-219 on streptavidin-coated wells. Detection was performed with primary anti-tau(pT212) antibody and secondary goat anti-rabbit antibody coupled to HRP. The graph is representative of two experiments. E, Detection of phosphorylated tau207-219 in the presence of excess unphosphorylated peptide. Different amounts of phosphorylated tau207-219 (0.01 pmol, 0.1 pmol, 1 pmol) were mixed with a dilution series of unphosphorylated tau207-219 (12.5 - 100 pmol). Signals obtained in wells loaded only with the same amount of the unphosphorylated peptide were subtracted from the read-out of the mixtures. The graph is representative of two experiments. In B-E, error bars indicate the difference between duplicate wells.

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