Aug 02, 2023

Public workspacesample_prep_serum.nan V.1

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DOI
dx.doi.org/10.17504/protocols.io.6qpvr34epvmk/v1
  • 1Network for Advanced NMR (NAN);
  • 2University of Georgia
  • Christopher Esselman: Protocol review;
  • Leandro I Ponce: Protocol review
NAN support at UGA
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr34epvmk/v1
Protocol Citation: NAN KB, Mario Uchimiya, John Glushka, Christopher Esselman, Leandro I Ponce, Laura Morris, Arthur Edison 2023. sample_prep_serum.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr34epvmk/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: August 02, 2023
Last Modified: August 02, 2023
Protocol Integer ID: 85858
Funders Acknowledgement:
National Science Foundation
Grant ID: 194670
Disclaimer
This protocol is developed and maintained by Network for Advanced NMR (NAN). The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to this protocol is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with this protocol, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This is a modified protocol for a protein precipitation method for plasma/serum samples. This protocol was originally proposed by:
CITATION
Nagana Gowda GA, Raftery D (2014). Quantitating metabolites in protein precipitated serum using NMR spectroscopy.. Analytical chemistry.
LINK
https://doi.org/10.1021/ac5005103
See also:
CITATION
Beckonert O, Keun HC, Ebbels TM, Bundy J, Holmes E, Lindon JC, Nicholson JK (2007). Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.. Nature protocols.

CITATION
Nagana Gowda GA, Raftery D (2019). Analysis of Plasma, Serum, and Whole Blood Metabolites Using 1H NMR Spectroscopy.. Methods in molecular biology (Clifton, N.J.).
LINK
https://doi.org/10.1007/978-1-4939-9690-2_2

Materials
1. Chemicals and reagents
  • Phosphate buffer in D2O (100 mM, pH 7.4, 1/3 mM DSS-d6)
  • Methanol

2. Equipment
  • Calibrated micropipettes (100 μL, 200 μL, and 1000 μL)
  • Pippette tips
  • 1.5-mL Eppendorf tubes
  • 5-mm SampleJet NMR tubes (Bruker)
  • Centrifuge
  • Vortex mixer
  • Speed-vac concentrator
Day-1/2
Thaw samples TemperatureOn ice or at Temperature4 °C

Add Amount600 µL of 100% cold methanol to Amount300 µL of samples TemperatureOn ice
  • Use 1.5-mL Eppendorf tubes
  • Keep methanol cold TemperatureOn ice

Pipetting
Critical
Vortex the samples for Duration00:00:10

10s
Incubate the samples at Temperature-20 °C for Duration00:20:00

20m
Centrifuge the samples at Temperature4 °C at Shaker16000 rcf for Duration00:30:00

30m
Centrifigation
Transfer the supernatants to new 1.5-mL Eppendorf tubes
Dry the samples in a speed-vac concentrator DurationOvernight (time varies depending on the sample condition)

Note
Store dried samples at Temperature-80 °C until the following step if needed


Overnight
Day-2/2
Thaw the samples TemperatureOn ice or in Temperature4 °C
Optional
Add Amount600 µL of the phosphate buffer to each sample

Pipetting
Critical
Vortex the samples at Temperature4 °C for Duration00:10:00
10m
Centrifuge the samples at Temperature4 °C for Duration00:00:10
10s
Centrifigation
Transfer Amount580 µL to 5-mm NMR tubes

Note
No stickers/labels on the caps and tubes allowed

Citations
Nagana Gowda GA, Raftery D. Quantitating metabolites in protein precipitated serum using NMR spectroscopy.
https://doi.org/10.1021/ac5005103
Beckonert O, Keun HC, Ebbels TM, Bundy J, Holmes E, Lindon JC, Nicholson JK. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.
Nagana Gowda GA, Raftery D. Analysis of Plasma, Serum, and Whole Blood Metabolites Using 1H NMR Spectroscopy.
https://doi.org/10.1007/978-1-4939-9690-2_2