Histological damage scores were significantly higher in the AIA group compared to the naive group

o recognize GST-tagged recombinant GCK with macrocyclic europium attached to an anti-GST antibody, and XL 665 attached to an anti-FLAG antibody was Rutin web utilized to recognize FLAG-tagged recombinant GKRP, initially in 384-well-plate format. The assay was validated using a range of GCK and GKRP concentrations at manufacturer-recommended detection antibody concentrations. Fluorescence was measured immediately upon mixing, and every 30 minutes thereafter for a total of 240 minutes. The plate was covered and incubated at room temperature throughout the course of the experiment. The signal reached a maximum at t = 60 minutes and remained stable; the results at 60 minutes are presented in The signal ratio in the absence of S6P in this experiment was similar to that observed in 384-well format at 5.56 nM of both proteins. The S6P EC50 value given from a nonlinear regression of these data was 0.460.08 mM, in agreement with previous findings. As S6P enhanced detection and is available commercially for a fraction of the cost of GKRP, subsequent experiments were run including S6P at its EC80 value. The EC80 value represents a compromise between the desire for large enough signal enhancement and the need to maintain a binding level that is not too saturated so that it can be outcompeted by compounds of interest. Additionally, this approach enables detection of specific competitors of S6P, which would likely be acting on GKRP as desired, and not on GCK. Therefore, the effect of known inhibitors of the GCK-GKRP interaction, which should decrease the fluorescence signal, were tested at 5 nM GCK, 5 nM GKRP, and 2 mM S6P. Four compounds were tested at five half-log concentrations below and above their predicted half-maximal inhibitory concentration values: glucose, F1P, and two commercially available GKAs. Both glucose and GKAs interfere with the binding of GKRP to GCK and decrease GKRP inhibition, while F1P binds to GKRP and is competitive with respect to S6P. All four compounds appreciably inhibited the GCK-GKRP interaction with an associated decrease in the HTRF signal . Therefore, concentrations of 5 nM GCK, 5 nM GKRP, and 2 mM S6P were suitable for detection of inhibition of the GCK-GKRP interaction via both GCK and 5 GCK/GKRP Assays tag detection reagents were added following a pre-read to detect compound autofluorescence. The assay performed well across all plates , and demonstrated consistent inhibition by the control compound GKA-EMD delivered as a titration within each screening plate. Additionally, there was no PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 loss in assay performance upon overnight storage of the reagents on ice. Counter-assay to Identify Compounds Interfering with HTRF Detection The HTRF-based assay was adapted to detect small molecules that interfere non-specifically with protein-protein interactions, GCK/GKRP Assays with HTRF reaction components, or with detection conditions. A rabbit anti-mouse IgG antibody was utilized as the FRET donor in place of the GST K antibody, with the monoclonal mouse antiFLAG XL 665 antibody utilized as the FRET acceptor. Accordingly, mixture of these two PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19637192 antibodies should result in donor-acceptor antibody interaction, and thus detectable FRET signal, in the absence of interference. True positives, such as F1P and S6P, should show no concentration-dependent change in HTRF signal in this control experiment, while compounds that show fluorescent liabilities in either the donor or acceptor channels should result in a change in signal in this IgGbase