SAMPLES

Types of Samples

The NMR facility was designed to deal primarily with Biological Macromolecules. Consequently we do not have the capabilities to handle explosive, pressurized, chemically corrosive, radioactive, biologically dangerous or otherwise hazardous samples. If you have a sample that falls into one of these categories you should discuss your requirements with the Facility Manager before starting any experiments. However this does not absolve any user from responsibility for whatever harm or damage that such samples may cause to the Facility.

The Facility was designed for Solution NMR studies, primarily aqueous solutions. We do not have the capabilities to study solid samples.

NMR Tubes

NMR tubes can be purchased from the Wilmad Glass Company for routine use we recommend 5mm 528-PP 7, for better results we suggest 5mm 535-PP 7 tubes. If sample volume is limited users should consider buying low volume samples tubes from Shigemi, Inc (412-444-3011), (Again 5mm outer dia). Users should make sure that the tubes are matched to the solvent that they are using.

Sample Preparation

The volume of the samples should be at least 500 microliters and contain at least 10% deuterium oxide and a chemical shift standard. If the sample is in a buffer the buffer salt shoud be deuterated. Deuterated solvents, buffers and chemical shift standards are available from almost any chemical supplier.

For studies of secondary and tertiary structure of biomolecules (eg. protein) the sampel should be >90% purity and in concentrations of >1 mM. Higher purities and concentrations are recommended to obtain better NMR spectra. For long term preservation of the sample it should contain

The size, purity and sequence of purified biomolecules should be verified by mass spectrometry, amino acid analysis and amino acid sequencing and/or DNA sequencing.

Dynamic light scattering and/or sedimentation ultracentrifugation can be used to verify the oligomeric state at NMR sample protein concentrations.

Three-dimensional structures of folded polypeptides containing 35-70 amino acids can be studied by homonuclear (proton) NMR. The structures of monomers under 25 kD and symmetric multimers under 40 kD have been solved using heteronuclear NMR approaches, but require nitrogen-15, carbon-13 and/or deuterium labeling. Isotope labeled protein is typically produced in E. coli using isotope labeled growth media this is available from Isotec, Cambridge Isotope Laboratories, and Martek

Some useful references:

Chemical Shift Standards:
David S. Wishart et al. (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR, Journal of Biomolecular NMR 6, 135-140.

NMR Buffers:
Catherine H. Schein (1990) Solubility as a function of protein structure and solvent components, Biotechnology 8, 308-316.

Jens Freund and Hans R. Kalbitzer (1995) Physiological buffers for NMR spectroscopy, Journal of Biomolecular NMR 5, 321-322.

General NMR:
Three Methods in Enzymology volumes (176, 177 and 239) are dedicated to biomolecular NMR. See Norman Oppenheimer's article "Sample Preparation" pp 78-89, Vol 176 for useful hints on preparing a NMR sample.