Serum Preparation for 1D NMR

1. Serum sample are first aliquoted

2. Proteins are precipitated by the addition of twice the amount of methanol as sample

3. Antibacterial NaN3 is added 2.0% by volume to avoid any bacterial growth

4. The 1:2 mixture of serum: solvent is then vortexed for 10 seconds

5. Samples are incubated at 4°C for 10 minutes

6. Next, the samples are centrifuged at 14,000 rpm for 20 mins at 4°C to pellet the proteins

7. The supernatant is collected and centrifuged at 14,000 rpm for 5 min a 4°C to pellet any leftover protein

8. At this point, the supernatant samples are collected and snap frozen in Liquid Nitrogen and freeze dried to remove methanol by speed vacuum centrifugation (SpeedVac R Plus, Savant) and water by lyophilization using FreeZone™ (Labconco, Kansas City, MO) for at least 24 hours and stored at -20°C until NMR analysis.