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The basic principle of the approach is that a fluorescence pattern of a mixed sample is a superposition of the fluorescent patterns for each target.
CaMKII is activated by Ca2+/CaM and ATP in one mixed sample (blue squares) while the other mixed sample is unactivated (red circles).
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The mixed sample was pressed homogeneously into the sample holder of the oven with a thickness of 2 mm, which is sealed using graphite foil with a thickness of 125 μm on both sides.
Then, each mixed sample was digested with Trypsin Gold (Promega, Madison, WI, USA) at 37 °C for 16 hours and labeled according to the manufacturer's protocol for the 8-plex iTRAQ reagent (Applied Biosystems).
Then, 50 g of the mixed sample was transferred into the enrichment medium.
On the other hand, novel mass peaks only observed in the mixed sample were discovered and may serve as biomarkers for mixtures or contaminations [83, 86].
The mixed sample was centrifuged (10,000×g at 4°C) for 5 min to obtain the RNA pellet.
A volume of 25 µl (corresponding to ∼13 µl of plasma) of the mixed sample was then transferred onto filter paper and left to dry overnight.
The 10 μL mixed sample was used for 12% SDS-PAGE loading.
Five minutes later, the absorbance at 545 nm of the final mixed sample was measured.
This mixed sample was applied via anodic cup loading on one gel strip.
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