The Orexin 1 Receptor (OX1R) may be an important therapeutic target for treatment of sleep disorders, obesity, emotional stress, and addiction. The OX1R Reporter Assay Kit consists of a 96-well plate coated with a transfection complex containing DNA constructs for OX1R, a recombinant G-protein, and a cAMP response element regulated Secreted Alkaline Phosphatase (SEAP) reporter (OX1R Reverse Transfection Strip Plate). Cells grown on the transfection complex will express OX1R at the cell surface and the recombinant G-protein inside the cell. Binding of agonists to OX1R initiates a signal transduction cascade resulting in expression of SEAP which is secreted into the cell culture medium. Aliquots of culture medium are removed at time intervals beginning at about 16 hours and SEAP activity is measured following addition of a luminescence-based alkaline phosphatase substrate provided in the kit. The kit is easy to use and can be easily adapted to high throughput screening for therapeutic compounds regulating activation of OX1R. A known OX1R agonist, Orexin A, is included in the kit for use as a positive control. The kit provides sufficient reagent to measure SEAP activity at three time points, using the black plates provided.
Orexin 1 Receptor Reverse Transfection Strip Plate: 1 plate; -20°COrexin A Positive Control: 1 vial/10 µl; -80°CSEAP Substrate (Luminescence): 1 vial/15 ml; 4°C96-Well Solid Plate (black) with lid: 3 plates; Room Temperature