Biomolecular NMR Sample Preparation Tips

Sample Quantity

The minimum concentration/quantity of material required to prepare an NMR sample is large by biochemical standards:

  • For 1D work, at least 100 µM (0.5 mg).
  • For multi‐D work, at least 1 mM (5 mg).

These estimates are based on a 10 kDa molecule in 500 µL solution.

Purification of sufficient quantities of biomolecules at mM concentrations is not the only difficulty. At high concentrations, unfavorable aggregation or loss of solubility often occurs. Experiment with pH and salt concentration to obtain a stable solution.

Sample Tubes

Use good quality NMR sample tubes (e.g. Wilmad 528 tubes) to obtain optimal resolution. These tubes require about 600 µL of sample (about 40% of which is outside the detection volume of the receiver coil).

The sample volume can be reduced to about 350 µL using one of the following approaches:

Wash tubes with a detergent solution and rinse thoroughly with distilled, deionized water. Avoid the use of strong acids containing metal ions and of organic solvents.

Tubes may be dried by blowing with dry N2 or if absolutely necessary in a clean oven at < 50°C. Exceeding 50°C will ruin the tube!


Non‐aqueous: Use 100% deuterated solvent (e.g., DMSO‐d6) to avoid a large solvent background signal. Keep the tube and the solvent dry, avoid exposure to air. If your sample is dilute (< 5 mM) it is a good idea to prepare a second sample tube containing just the solvent so that you can subtract the signals arising from solvent impurities.

Aqueous: When possible use 100% D20. However, you will usually have to use non‐deuterated H2O in order to observe labile protons (e.g., amide protons in proteins). In this case use 90% H2O/10% D2O, so that there is a sufficient deuterium signal for locking.

Buffer solutions should not contain paramagnetic ions and should have a total salt concentration of less than 200 mM. Any organic component of the buffer solution (e.g., Tris, acetate, etc.) in excess of 500 µM should be deuterated to avoid background signals.

If accurate chemical shifts are required an internal standard should be used. For organic solutions use tetramethylsilane (TMS), for aqueous solutions 3,3,3‐trimethylsilylpropionate (TSP), or 5,5‐dimethylsilapentanesulfonate (DSS).

Suppression of Bacterial Growth

Proteases and nucleases excreted by bacteria will degrade your sample over time. If you have problems with sample degradation, the following may help:

  • Bacteria are effectively suppressed in > 80% D2O.
  • Use a chelator (e.g., EDTA, EGTA) to complex divalent cations.
  • Add NaN3 (< 50 µM) to your buffer.
  • Use microporous filtration as last step in your prep.

Paramagnetic Impurities

Paramagnetic ions (e.g., Fe3+, Mn2+) cause large distortions in chemical shifts and relaxation times if they form complexes with your molecule. Solvated paramagnetic ions will also cause broadening of the water signal, which will severely compromise solvent suppression. Buffer solutions must not contain these ions, however, they are often found in small concentrations as impurities.

Avoid the use of chromic acid or other metal‐containing oxidants for tube cleaning. Avoid contact between your solution and stainless steel (e.g. in HPLC plumbing). To remedy any problems, add 50 µM EDTA to sequester paramagnetic ions or remove paramagnetics by incubating with Chelex.