There was a problem with using the kit FFAR1 Reporter Assay Kit (Cayman Chemical). The kit is based on transfection of cells HEK293T with a DNA complex of FFAR1, altered G-protein, and secreted alkaline phosphatase (SEAP). When exposed to receptor agonists, the cells produce SEAP and its activity is measured by adding a luminescence-based substrate. Before analysis, incubation (30 min 65C) is performed to neutralize the endogenous alkaline phosphatase.
The first time the experiment was successful, but an attempt to repeat it after some time failed. Only the medium with reduced serum changed. The luminescence of the control samples did not differ from the positive control included in the kit (GW9508). During new attempts, it was found that incubation to inactivate alkaline phosphatase has no effect on luminescence. That is, luminescence values are similar before and after incubation, or even slightly increased. Changing the incubation parameters did not change anything. What can be done to resume successful experiments on this kit?
I would be grateful for any suggestions.
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