Orexin A Analysis in Plasma
daniel.dautan daniel, Wojciech Paslawski, Per Svenningsson
Abstract
Mouse plasma analysis of Orexin A using Novus Biologicals ELISA kit (NBP2-80231).
Steps
Centrifuge samples for 0h 15m 0s at 1000×g at 4°C.
Add50µL of standards' working solutions and samples to 96-well plate in duplicates.
Add 50µL of biotinylated detection antibody working solution to each well. Cover with the provided plate sealer and incubate for 0h 45m 0s at 37°C.
Decant the solution from each well. Add350µL of wash buffer added to each well and allowed to soak for 0h 1m 0s.
Decant the solution from each well and pat dry against clean absorbent paper. Repeat this wash step (steps 4-5) 3 times.
Add 100µLof HRP conjugate working solution to each well. Cover plate with the sealer and incubate for 0h 30m 0s at 37°C.
The wash step was performed as described above. Add 90µL of substrate reagent to each well. Incubate for 0h 15m 0s at 37°Cprotected from light.
Add 50µL of stop solution to each well. Measure the optical density using a micro-plate reader
with absorbance set to 450 nm.
