The reference exposure is designed to initially drive the photopigment system to an identical state of photoequilibrium at the beginning of each series of stimulations, prior to the adapting stimulus that drives the responsiveness of the system to a new state, assayed during the test stimulation [8], [9].
GTPase activities of purified proteins were subsequently estimated in steady state assays using the malachite green assay (BioAssay Systems).
Although the Y96L mutant has a substantially reduced activity in steady state assays, the activity does increase with an increasing mutant Cdx concentration, indicating that the role of Tyr96 is primarily in binding and not electron transfer.
Pre-steady state assays, such as that in Figure 3D, were carried out in a similar way using nucleotide-free MCAK (produced as described below) at a concentration of at least 1 μM and with 3′mant-2′deoxyATP in at least 10-fold excess over the concentration of MCAK.
The binding and uptake of TfR was kinetically analyzed over 2 hrs with saturating levels of A568Tf (Figure 3A) in an approach to steady-state assay (Materials and Methods; [32]).
Blood samples were obtained before and after filtering, and urine samples and ultradiafiltrate samples were also collected for a steady-state assay of micafungin.
A closer look at our progress curves, however, did not reveal lag or burst phases in the time scale of our steady-state assay conditions.
The ATP biosensor was tested in a steady-state assay in which ATP was produced from ADP and phosphoenolpyruvate, catalyzed by pyruvate kinase.
To demonstrate its ability in a different type of assay, Rho-MatB was also tested in a steady-state assay in which ATP was consumed by PcrA helicase, coupling ATP hydrolysis to translocation on DNA.
The mRNA levels of the wild-type luciferase reporter in the mRNA steady-state assay do not differ when the reporter is expressed in wild-type and upf1Δ cells, rather these values are within error of each other (amount of luciferase mRNA in cell-line/luciferase mRNA in wt cells = 1 ± 0.28, for wt, and 0.75 ± 0.16, for upf1Δ).
Mixing of Dpo4 mutant Y108W with DNA in the absence of the CPM acceptor did not result in a detectable time-dependent change in fluorescence, indicating that the quenching of Trp fluorescence observed in the steady-state assay occurred within the dead time (∼1 ms) of the stopped-flow apparatus, as would be expected for quenching due to inner filter effects.
