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Validation of research methodology is critical in research design.Correlation between experimental observables must be established beforeundertaking extensive experiments or propose mechanisms. This article showsthat, observables in the popular calcium flux strength assay used in thecharacterization of sweetener-sweet taste receptor (STR) interaction areuncorrelated. In pursuit to find potential sweeteners and enhancers, calciumflux generated via G-protein coupling for wildtype and mutant receptorsexpressed on cell surface is measured to identify and localize sweetenerbinding sites. Results are channeled for sweetener development with directimpact on public health.  We show that flux strength is independent of EC50 and sweet potency. Sweetpotency-EC50 relation is non-linear and anti-correlated. Single point mutantsaffecting receptor efficiency, without significant shift in EC50 have beenpublished, indicating flux strength is independent of ligand binding. G-proteincoupling step is likely observed in the assay. Thus, years have been spentgenerating uncorrelated data. Data from uncorrelated observables does not givemeaningful results. Still, majority of research in the field, uses change incalcium flux strength to study the receptor. Methodology away from fluxstrength monitor is required for sweetener development, reestablish bindinglocalization of sweeteners established by flux strength method. This articleserves to remind researchers to validate methodology before plunging into longterm projects. Ignoring validation test on methodology, have been a costlymistake in the field. Concepts discussed here is applicable, wheneverobservable in biological systems are many steps moved from the event ofinterest.

Importance of method validation: Implications of non-correlated observables in sweet taste receptor studies