May 22, 2024
Orexin A Analysis in Plasma
- daniel.dautan daniel1,
- Per Svenningsson1,
- Wojciech Paslawski1
- 1Karolinska Institute Stockholm
Protocol Citation: daniel.dautan daniel, Per Svenningsson, Wojciech Paslawski 2024. Orexin A Analysis in Plasma. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx365zg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 15, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95308
Keywords: ASAPCRN, Serum
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
Mouse plasma analysis of Orexin A using Novus Biologicals ELISA kit (NBP2-80231).
Materials
ELISA Orexin A kit- Novus Biologicals, NBP2-80231, MO, US
Centrifuge samples for 00:15:00 at 1000×g at 4 °C .
15m
Add50 µL of standards' working solutions and samples to 96-well plate in duplicates.
Add 50 µL of biotinylated detection antibody working solution to each well. Cover with the provided plate sealer and incubate for 00:45:00 at 37 °C .
45m
Decant the solution from each well. Add350 µL of wash buffer added to each well and allowed to soak for 00:01:00 .
1m
Decant the solution from each well and pat dry against clean absorbent paper. Repeat this wash step (steps 4-5) 3 times.
Add 100 µL of HRP conjugate working solution to each well. Cover plate with the sealer and incubate for 00:30:00 at 37 °C .
30m
The wash step was performed as described above. Add 90 µL of substrate reagent to each well. Incubate for 00:15:00 at 37 °C protected from light.
15m
Add 50 µL of stop solution to each well. Measure the optical density using a micro-plate reader
with absorbance set to 450 nm.