Biomolecular NMR Sample Preparation

Sample Quantity

The minimum 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).

In practice, purifying a sufficient quantity of material is not the only difficulty. At millimolar concentrations many biomolecules have unfavourable aggregation or solubility properties. You may have to experiment with pH and salt concentration to obtain a stable solution.

Sample Tubes

Use good quality NMR sample tubes (e.g., Wilmad 528-PP, available at ISU Chem. Stores), to obtain optimal resolution. These tubes require 600 µL of sample solution, of which about 40% is outside the detection volume of the receiver coil.

Alternatives (to reduce the minimum sample volume to 350 µL):

• Shigemi tubes (expensive, but then so are samples)
• Doty susceptibility plugs, available from Wilmad (also expensive)

Wash tubes with a detergent solution (e.g., Contrad). Rinse thoroughly with distilled, de-ionized water.

Avoid:

• Strong acids containing metal ions.
• Organic solvents.

Tubes may be dried:

• by blowing with dry N₂
• 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 the 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 undeuterated 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 the lock system to function.

Buffer solutions should not contain paramagnetic ions and should have a total salt concentration < 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.

For work where accurate measurement of chemical shifts is needed an internal standard should be used:

• Organic solutions: tetramethylsilane (TMS).
• Aqueous solutions: 3,3,3-trimethylsilylpropionate (TSP), or 5,5-dimethylsilapentanesulfonate (DSS)

Suppression of bacterial growth

Bacteria excrete proteases and nucleases that 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 wll severely compromise solvent suppression. Buffer solutions must not contain these ions, however, they are often found in small concentrations as impurities.

Things to avoid:

• Don't use chromic acid or other metal-containing oxidants for tube cleaning.
• Avoid contact between your solution and stainless steel (e.g. in HPLC plumbing.)

• Add 50 µM EDTA to sequester paramagnetic ions.
• Remove paramagnetics by incubating with Chelex.