PCR reactions were carried out by triplicate using fixed amounts of template DNA.
Drug interaction analyses described above were performed using fixed amounts of antibodies and TNF-α.
Using fixed amounts of SR ΔRS2), we found that the addition of SR RS2) to SRPK1 ΔN) or SRPK1 ΔS), unlike the wild-type kinase, caused no activation of SR ΔRS2) phosphorylation.
Competition reactions were carried out using fixed amounts of SR ΔRS2) or SRSF1 (50 nM) as a substrate and varying concentrations of the competitor SR RS2).
The activation constant (KA) for SR RS2) to SRPK1 using fixed amounts of SR ΔRS2) ([S]) was determined using eq 3: 3where γ is the change in kcat for SR ΔRS2) phosphorylation in the presence of SR RS2) in the activation site, KSR is the Km for SR ΔRS2), and [A] is the concentration of SR RS2).
The dissociation constant (KI) for SR RS2) to SRPK1 using fixed amounts of SRSF1 was measured using eq 2: 2where vi/ vo is the relative initial velocity (ratio of v in the presence and absence of inhibitor), KSR is the Km for SRSF1, and [I] is the SR RS2) concentration.
The Supplemental Figure S1 data will be very disturbing, if one's only experience with intercalators has been to use a fixed amount of intercalator to quantitate an unknown amount of duplex DNA.
Since a significant fraction of genes do not have experimentally supported 3' UTRs, some algorithms simply use a fixed amount of genomic sequence downstream from the stop codon as the 3' UTR.
To determine if in this situation NEDD8 could still be activated, we next performed an in vitro competition assay using thioester formation as readout with fixed amounts of UBE1 (100 nM) and ubiquitin (0.8 μM) and increasing amounts of NEDD8.
One program, T.A.F., allowed banks and investment firms to compete in auctions for fixed amounts of Fed funding, while T.S.L.F.
