However, when imidazole, which competes with histidine for Ni-NTA binding, was added to the supernatants, histags of the trimer protein were again detected by ELISA.
One hundred microlitres of rabbit anti-bovine IgA horseradish peroxidase (HRP) conjugate (Bethyl Laboratories, Inc. USA), diluted 1 10,000 in conjugate diluent containing 4 % horse serum (to prevent non-specific binding) was added to each well and the plates were again incubated for 1 h at 37 °C followed by washing as previously described.
However, no environmental bindings are added to the devices in these approaches; thus, the use of devices is only based on their intrinsic characteristics without taking the context of the patient into account.
In brief, 100X unlabelled DNA competitors to 70 well-characterized DNA-binding proteins were added to the binding reaction described above, using arrays of 10 competitors per reaction, with a 30-min incubation on ice prior to the addition of labelled probe.
Before flow cytometric analysis, 400 μl of 1× binding buffer were added to the cells and the extent of apoptosis was determined by analyzing 15,000 ungated cells using a FACScan flow cytometer and Cell Quest software (FACSCalibur; Becton-Dickinson, San Jose, CA).
Then 400 µl of 1× binding buffer were added to each sample which was analyzed by flow cytometry to quantify necrotic and apoptotic cells.
Subsequently 150 µl of 1x Annexin-V antibodies in binding solution were added to each well, followed by 15 min incubation.
Six tandem copies of a synthetic miR-312 binding site were added to the 3' UTR of a UAS-GFP transgene (Fig. 1C).
In all, 450 μl of binding buffer were added to terminate the reaction.
Finally, 400 μl of 1× binding buffer were added to each tube and the samples were analyzed by flow cytometry within 1 hour.
