Skip to main content
Center for Biomolecular NMR Spectroscopy
HomeUser GuidelinesSpectrometersSchedulingFeesUNIX Software InformationEducation and USER BASELinksContact Us
Buffer Exchange Procedure for the Preparation of NMR Samples

Bar Break


The preparation of a biomolecule for NMR studies usually requires exchanging the sample in an aqueous solution appropriate for conducting the proposed NMR studies. The two main requirements that must be met to successfully acheive this include:

1. Useage of buffer components lacking covalently attached protons. This is important as the buffer will typically be present at much higher concentrations than that of the biomolecule, thus causing the NMR signals of the buffer to interfere with those of the biomolecule.

2. Inclusion of deuterated water (D2O) at a level of 5 % or greater. This is necessary as the stability of the NMR spectrometer is maintained by continuously monitoring the deuterium signal from the solvent.

There are two other important points to keep in mind. The first is that the total ionic strength should be kept as low as possible (compatible with the solubility/stability of your particular biomolecule). This is important as the coupling of the RF signal in the coil to the sample will be more efficient as the ionic strength is lowered. The second is that the pH should be kept low (below 6.5) if possible. This is because the exchange of the backbone amide proton is base-catalyzed; thus its exchange rate increases as the pH is raised. This can be bad because if the exchange rate becomes too rapid, the amide proton will exchange during the course of the nmr pulse sequence, and therefore the amide group will not be detected.

Although there are many ways to prepare samples of biomolecules in aqueous solutions containing 5% D2O, the following is one that I have been found to be particularly simple, reliable, and fast!
1. Transfer your biomolecule to standard dialysis tubing and dialyze against your final desired NMR buffer (e.g. 25 mM dibasic sodium phosphate, 25 mM NaCl, 5% D2O, pH 6.0 (as a guide to figure out the number of times to dialyze and the volume, simply calculate the cumulative effective dilution factor necessary to bring down any contaminating species to 0.1 times the protein concentration).

2. Wash a centricon unit (these are one-time centrifugal concentrators sold by Millipore; various molecular weight cutoff membranes are available, e.g. 3k, 10k, 30k, ..) using an aliquot of your nmr buffer (rinse once, discard, refill, and then centrifuge for 5 mins). Transfer your protein solution and concentrate to about 150 - 200 uL. Importantly, use the cap supplied, not parafilm, as the parafilm will create an airtight seal; this can be a problem as a chilled centrifuge will cause a vacuum to form in your sample; when removing the parafilm, this can cause the membrane to be lifted out of the o-ring that holds it in place, causing your sample to spill out the bottom!

3. Remove the NMR sample from the centricon (by inverting it and by centrifuging). Transfer to a 5 mm Shigemi NMR tube. Rinse the centricon with NMR buffer (once or twice) so that the final volume of the NMR sample is 320 uL.

4. Seal the NMR tube first with teflon tape and then with parafilm.


In order to facilitate the preparation of NMR samples, UTHSCSA general stores now carries a) 99.9% D2O and b) 5 mm Shigemi tubes for Bruker probes (as of July, 2003). The D2O is sold in 100 mL bottles and the order ID is 175013788. The Shigemi tubes are sold in a quantity of one and the order ID is 175042166. If you need larger quantities, you might contact one of the stable isotope suppliers or Shigemi directly.

Bar Break

Home  -  Getting Started  -  Spectrometers  -  Scheduling  -  Fees  -  UNIX Information  -  Technical Resources  -  Links  -  Contact
UTHSCSA  -  Center for Biomolecular Structure Analysis  -  Department of Biochemistry  -  Disclaimer  -  Copyright

Last updated on January 26, 2004