May 22, 2024

Public workspaceMelatonin ELISA

DOI
dx.doi.org/10.17504/protocols.io.81wgbx35ylpk/v1
  • 1Karolinska Institute Stockholm
Eileen Ruth Torres
Open access
DOI: dx.doi.org/10.17504/protocols.io.81wgbx35ylpk/v1
Protocol Citation: daniel.dautan daniel, Per Svenningsson 2024. Melatonin ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx35ylpk/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: 95309
Keywords: ASAPCRN, melatonin, sleep
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
Measurement of mouse plasma melatonin using ELISA Kit (Enzo Life Sciences, ENZ-KIT150-0001, NY, US) according to manufacturer instructions.
Materials
Enzo Life Sciences, ENZ-KIT150-0001
Melatonin Extraction
Melatonin Extraction
Mix Amount200 µL of plasma with an equal volume of cold Ethyl Acetate. Vortex gently.
Allow layers to separate on ice for Duration00:03:00 . Vortex again and incubated TemperatureOn ice for Duration00:02:00 .
5m
Spin samples at 1000g for Duration00:10:00 at Temperature4 °C . Transfer the organic layer to a new tube.
10m
Dry samples and resuspend in Amount220 µL of 1X stabilizer.
ELISA
ELISA
2h
Add Amount100 µL of standards' working solutions and samples to provided 96-well plate in duplicates.
Immediately after add Amount50 µL of melatonin tracer to each well (except blanks) followed by Amount50 µL of 1X melatonin antibody (except blanks).

Cover plates with the provided plate sealer. Incubate for Duration01:00:00 at Temperature37 °C with 500 rpm shaking.
1h
Decant the solution from each well. Add Amount400 µL of wash solution to each well.
Decant the solution from each well and pat dry against clean absorbent paper.
Repeat wash step 3 times.
AddAmount200 µL of melatonin conjugate solution to each well (except blanks). Cover plate with the sealer. Incubate for Duration00:30:00 at TemperatureRoom temperature .

30m
Perform the wash step as described above. Add Amount200 µL of substrate reagent to each well. Incubate for Duration00:30:00 at Temperature37 °C protected from light.

30m
Add Amount50 µL of stop solution to each well. Measure the optical density using a micro-plate reader with absorbance set to 450 nm.