Two different acceptor beads can be used: with AlphaScreen acceptor beads, final emission is from rubrene (λem=520–620 nm); with AlphaLISA acceptor beads, the final emission is from europium that is doped into the beads which shows a much narrower emission spectrum (λem=615 nm). For protein kinases,

typically a biotinylated peptide substrate is conjugated to streptavidin coated donor beads and a phosphospecific antibody is conjugated to protein A coated acceptor bead ( Von Leoprechting et al., 2004). Because long wavelength light is used for excitation and emission occurs at shorter wavelengths, optical interference of the excitation light by compounds

is reduced. However, AlphaScreen has been shown to be susceptible to compound interference by color quenchers of the emission light as well as anti-oxidants, singlet oxygen http://www.selleckchem.com/products/cx-5461.html quenchers, Proteasome inhibitor and biotin mimetics if streptavidin coated beads are used ( Baell and Holloway, 2010). Another consideration in developing AlphaScreen assays is that variation of the biotinylated peptide substrate will show a “hook-effect” (as mentioned above) at high substrate concentrations due to saturation of the streptavidin donor bead binding sites – the signal first increases with increasing peptide, then levels off and starts to decrease when excess peptide is used as the proportion of productive donor acceptor bead complexes decreases due to the excess peptide in the assay ( Quinn et al., 2010). Therefore, it is not possible to determine Km values for the peptide substrate using AlphaScreen as binding capacity of the beads limits the detectable range of substrate concentration. An advantage of this detection strategy is that the large distance dependence (~200 nm) allows the employment Fludarabine manufacturer of physiologically relevant substrates. Analytical approaches to kinase detection include microfluidic

systems for separation based on electrophoretic and electro-osmotic properties of the labeled species (Cohen et al., 1999). In one such technology platform (Caliper, PerkinElmer), optimization of the elution gradient on the UPLC system allows one to run kinase assays at extremely low substrate conversion rates (Wu et al., 2006). In this system, separation and quantification of substrate and product is provided and detection and interfering fluorescent compounds are readily flagged. With generic systems available that detect ATP/ADP conversion one could ask if there is any advantage to protein kinase assay systems that rely on detection of phosphorylated peptides, especially when peptide-based systems are oftentimes incapable of incorporating natural protein substrates.