Varying amounts of ATP were used in the absence or presence of PKA. Performance* (%) was calculated using D RFU. The optimal concentration of ATP was determined to be 50 µM. At 1 mM ATP, 70% of the signal is retained in a PKA assay. % Performance = ( D[ATP] / Dmax)*100 where D = RFU-Enzyme - RFU+enzyme
Phosphorylated calibrator peptide and non-phosphorylated substrate were mixed in various ratios and Sensor added. A calibration curve with a bi-phasic signature was generated. The first, steep portion of the curve appears at low phosphopeptide concentrations and the second, flat portion, at high concentration of phosphopeptide. In experiments in which phosphorylation is expected to cover both portions of the curve (eg: enzyme concentrations curves), or in which quantitative determination of phosphorylation is required, it is recommended that a calibration curve be included as part of the experiment. Once experimental parameters have been established within one portion of the curve, calibrations are no longer necessary.
Phosphorylated calibrator peptide and non-phosphorylated substrate were mixed in various ratios and Sensor added. A calibration curve with a biphasic signature is generated. The first, steep portion of the curve appears at low phosphopeptide concentrations and the second, flat portion, at high concentration of phosphopeptide.
This figure shows varying reactions with various amounts of enzyme monitored continuously over 90 min (e). The reaction was initiated with the addition of ATP at 8 min. Panel (f) shows an enzyme concentration curve generated using data extracted from the continuous monitor experiment at t= 68 min (60 min reaction time). This data is compared to an enzyme concentration monitored in EndPoint mode.
This figure shows reactions using varying amounts of p70S6 kinase that was added to cell lysates and monitored continuously over 90 min. The reaction was initiated with the addition of ATP at 10 min. The data indicates that endogenous ATP present in the cell lysates initiates enzymatic activity before additional ATP is added (c). Panel (d) shows an enzyme concentration curve using data extracted from the continuous monitor experiment at t= 70 min (60 min reaction time). This data is compared to results from an enzyme concentration curve performed in cell lysates and monitored in EndPoint mode.
Decreasing concentrations of p70S6 kinase were added to wells containing lysates. Substrate was added at a concentration of 1 µM. The enzymatic activity is shown as a ratio of 490 nm/600 nm. Limit of detection (LOD)= 3X stdev -mean RFU at 0 pM.
The assay was performed as described in the corresponding Detailed Protocol using 100 µM ATP, 1 µM substrate and a serial dilution of p70S6 kinase. The enzymatic activity is shown as a ratio of 490 nm/600 nm.
This figure shows reactions using varying amounts of enzyme monitored continuously over 90 min with substrate and ATP concentrations of 0.25 µM and 50 µM, respectively. The data indicates that high concentrations of enzyme associate to the Sensor and interfere with the binding of phosphorylated substrate (a). This effect can be corrected by "normalizing" the data based on the negative control. Panel (b) is identical to the data shown in (a) but has been "normalized" using the no Enzyme control at t= 8 min. This is only necessary if the concentration of enzyme used displays a significant offset from the negative control.
This figure shows p70S6 kinase reaction in the presence of varying amounts of Staurosporine, monitored continuously over 120 min. The reaction was initiated with the addition of ATP at 10 min.
The assay was performed as described in the corresponding Detailed Protocol using 10 µM ATP, 0.5 nM p70S6 kinase and a serial dilution of Staurosporine.
Decreasing concentrations of Staurosporine were prepared in Assay Buffer and combined with p70S6 kinase. Activity was measured as outlined in the corresponding Detailed Protocol.
Decreasing concentrations of Staurosporine were prepared in Assay Buffer. The final concentration of substrate was 1 µM. The IC50 value is higher than in cell-free assays due to the presence of undetermined amounts of endogenous ATP.
Decreasing concentrations of enzyme were mixed with Substrate in Assay Buffer containing 75 µM ATP. The enzymatic activity is shown as a ratio of the wavelengths of 490 nm/600 nm (e). Product formation from the data generated in (e) was calculated using a phosphopeptide calibrator curve constructed simultaneously with the enzyme concentration curve (f).
Decreasing concentrations of enzyme were mixed with substrate at a concentration of 2 µM in Assay Buffer containing 75 µM ATP. The enzymatic activity is shown as a function of fluorescence quench (a). Product formation from the data generated in (a) was calculated using a phosphopeptide calibrator curve constructed simultaneously with the enzyme concentration curve (b).
Mouse brain protein extract was serially diluted in Assay Buffer. Phosphorylated and nonphosphorylated peptides were added and % performance was calculated using D RFU. % Performance = (DRFU/Dmax)*100 where D = RFU0%phospho peptide - RFU100%phospho peptide
Four compounds that inhibited enzyme activity up to 70% were identified by ratiometric analysis of 490 nm/600 nm emission wavelengths (left panel). When analyzed in "quench mode" at 490 nm (right panel), only 2 inhibitors were identified and several colored compounds caused over-recovery of fluorescence signal. Compounds are black circles and colored compounds are red circles. The controls are: no enzyme and no inhibitor (blue) and enzyme and no inhibitor (green). Controls were included on the same plate as the compounds in a final DMSO concentration of 1%. The final concentration of compounds was 5 µM. Dotted lines represent 3X the standard deviation of the mean of positive and negative controls (n= 16). Hits were confirmed in follow-up studies.
The sequences shown in the table are a representative list of several quencher-labeled peptide substrates that have been used successfully in the TruLight™ assay. *Requires Post-Reaction Buffer
The TruLight assays are based on a proprietary technology utilizing microspheres that are coated with fluorescent polymers and a metal ion coordination complex. Peptide substrates are labeled with a quencher that will quench fluorescence when bound. When the peptide becomes phosphorylated, it binds the microsphere, resulting in fluorescence quenching and a decrease in the fluorescent signal.
Green-Excitation Spectrum; Blue-Emission at 0% phosphorylation; Red-Emission at 100% phosphorylation. When substrate is phosphorylated by a kinase, the fluorescence of the Sensor decreases at 490 nm and the energy transfer from the Sensor to the dye is evident as an increase in fluorescence. In this example, energy is transferred to Lissamine Rhodamine B (LRB) and monitored at 600 nm.
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