Simultaneous multiplexed in vitro detection of Syk and Lyn kinase activities. (A) In vitro Lyn assay luminescence emission spectra in the presence of both 5-FAM-SFAStide-A and SAStide-Cy5. (C) In vitro Syk assay luminescence emission spectra in the presence of both 5-FAM-SFAStide-A and SAStide-Cy5. (E) In vitro Lyn and Syk assay luminescence emission spectra the presence of both 5-FAM-SFAStide-A and SAStide-Cy5. (B, D, F) Quantification of 5-FAM-SFAStide-A signal and SAStide-Cy5 signal for each assay. The green and red boxes represent the approximate spectral regions represented in the Gaussian fitted curves used to integrate the signal (see supporting information S6 for more detail). Assays were performed in the presence of 2.5 µM 5-FAM-SFAStide-A, 12.5 µM SAStide-Cy5, Lyn, Syk or both kinases (15 nM), 100 µM ATP, 10 mM MgCl2 and 0.2 ng/µL BSA.