Apr 06, 2023
Determination of NM Concentration
- Xiqun Chen1,2
- 1Massachusetts General Hospital;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Xiqun Chen 2023. Determination of NM Concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj8wxxgk5/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: April 06, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 80139
Keywords: ASAPCRN
Funders Acknowledgement:
From cancer associations to altered immunity in the pathogenesis of Parkinson's disease
Grant ID: ASAP-000312
Abstract
This is the protocol for determining neuromelanin concentration and data.
Place SNpc tissue in a plastic tube and carefully ground it. Weigh 10 mg of tissue for each sample, and place itin a 5 mL glass tube.
In each tube, add 1.5 mL of pH 7.4 phosphate buffer (50mM), shake, and centrifuge at 9000 x g for 30 mins. Discard the supernatant.
Wash with phosphate buffer and repeat once more.
Add 1.5ml of Tris buffer (50 mM, pH 7.4) solution, containing sodium dodecyl sulfate (5 mg/ml) and 0.2 mg/ml proteinase K to the pellet of each sample. Incubate the pellet by shaking in this solution for 2 hours at 37ºC.
Centrifuge the suspension of pigment at 9000 x g for 30 minutes.
Wash the pellet with 1.5 ml of NaCl solution (9 mg/ml) and 1.5 ml of water. Centrifuge at 9000 x g for 30 minutes.
Dissolve the NM residue in 1 ml of 1M NaOH at 80ºC for 1 hour.
Centrifuge this solution and transfer the supernatant into a quartz cuvette, measure the absorbance at 350 nm.
To run calibration curves dissolve known amounts of NM (ranging from 1 – 30 µg) in 1 ml of 1 M NaOH at 80ºC for 1 hour.
NM value was the average from 2-3 replicates. The final values of NM concentrations are expressed as µg/mg dry tissue.
Protocol references
Zecca, L., Fariello, R., Riederer, P., Sulzer, D., Gatti, A., & Tampellini, D. (2002). The absolute concentration of nigral neuromelanin, assayed by a new sensitive method, increases throughout the life and is dramatically decreased in Parkinson's disease.FEBS letters,510(3), 216-220.
Cassidy, C. M., Zucca, F. A., Girgis, R. R., Baker, S. C., Weinstein, J. J., Sharp, M. E., ... & Horga, G. (2019). Neuromelanin-sensitive MRI as a noninvasive proxy measure of dopamine function in the human brain.Proceedings of the National Academy of Sciences,116(11), 5108-5117.