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All assays were conducted in black, 384-well microtitre plates (Corning Costar, Cat. No. 3654), except for Bombesin (6-14) and Urotensin II, which used a different Costar plate type (Cat. No. 3710). The total assay volume was 40 μL. The order of sequential addition of reagents was as follows:
1. Add 20 μL of inhibitor or buffer
2. Add 10 μL of fluorescent peptide
3. Add 10 μL of membrane preparation
To achieve appropriate final concentrations of reagents, the stock solutions for plate loading of fluorescent peptide and membrane preparation were 4x concentrates and that for inhibitor was 2x concentrates.
Results and Discussion
Figures 2 and 3 demonstrate Apelin and Urotensin II pharmacology as examples for the peptides listed in Table 3. Both radiometric and FP data are demonstrated.
In each case, the pharmacology was compared to radiometric filtration assays and similar pharmacology was observed. This data is provided in Table 3 for these peptides and for Vasopressin and Bombesin(6-14) also.
An example of a scatter plot for Vasopressin binding to the V1b receptor subtype is provided in Figure 4. The high number of replicate data allows for an accurate assessment of the Z factor, which in this case is 0.51, which meets the criteria for "an excellent assay" in relation to screening as described by Zhang et al.12 The separation band12 between the two distributions, assuming Gaussian behavior, is greater than 40 mP.
Table 4 c
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