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HTRF IP-One Assay Performed on the PHERAstar and RUBYstar Plate Readers

himeric constructs. There is a strong correlation with reference methods and no cross reactivity with 50 M of the following (phospho) inositides phosphates could be observed: Myo-inositol, PIP2, PIP3, IP2, IP3 and IP4.

In addition, for a direct performance comparison of BMG LABTECHs PHERAstar with the RUBYstar the IP-One assay was applied upon the 1321N1-CCK1 cell line together with the agonist CCK8-sulfated. The experiment was performed in 96-well plates resulting in very close EC50 values and a very good correlation regarding the %inhibition / basal curve (figure 3). Z' calculations at an agonist concentration equal to EC80 resulted in Z' > 0.78 for both plate readers.


But reaching beyond these expected results, the IP-One assay has already proved its worth in the field of screening for inverse agonist compounds. Looked at closely, and given its transitory characteristics, calcium measurement is often not sensitive enough to specifically detect the inhibition of the GPCR constitutive activities, whereas a modulation in the concentration of IP1 is perfectly able to show this. Using the IP-One assay therefore represents a tempting new alternative when investigating Gq-coupled receptors via the quantification of a new secondary messenger for this pathway, IP1. The messengers stability opens the possibility of the IP-One assays application to cases where other HTS technologies fail to provide a satisfactory solution such as in the detection of inverse agonist activities. The assay also profits from the special qualities of d2, one of the latest improvements implemented in HTRF technology. Given its performance, d2 is now included in all HTRF<
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