May 23, 2024

Melatonin ELISA

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbx35ylpk/v1
  • 1Department of Clinical Neuroscience, Karolinska Institutet, 171 76 Stockholm, Sweden;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Jacquelyn Haytayan
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DOI: https://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 16, 2024
Last Modified: September 24, 2024
Protocol  Integer ID: 95309
Keywords: ASAPCRN, melatonin, sleep, melatonin elisa measurement of mouse plasma melatonin, melatonin elisa measurement, mouse plasma melatonin, using elisa kit, elisa kit, mouse plasma
Funders Acknowledgements:
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
Mix 200 µL of plasma with an equal volume of cold Ethyl Acetate. Vortex gently.
Allow layers to separate on ice for 00:03:00 . Vortex again and incubated On ice for 00:02:00 .
5m
Spin samples at 1000g for 00:10:00 at 4 °C . Transfer the organic layer to a new tube.
10m
Dry samples and resuspend in 220 µL of 1X stabilizer.
ELISA
2h
Add 100 µL of standards' working solutions and samples to provided 96-well plate in duplicates.
Immediately after add 50 µL of melatonin tracer to each well (except blanks) followed by 50 µL of 1X melatonin antibody (except blanks).

Cover plates with the provided plate sealer. Incubate for 01:00:00 at 37 °C with 500 rpm shaking.
1h
Decant the solution from each well. Add 400 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.
Add200 µL of melatonin conjugate solution to each well (except blanks). Cover plate with the sealer. Incubate for 00:30:00 at Room temperature .

30m
Perform the wash step as described above. Add 200 µL of substrate reagent to each well. Incubate for 00:30:00 at 37 °C protected from light.

30m
Add 50 µL of stop solution to each well. Measure the optical density using a micro-plate reader with absorbance set to 450 nm.