Notes on sample requirements and isotope labelling

Improved hardware technology including cryogen-cooled probes, and new data acquisition approaches such as fast acquisition and non-uniform sampling mean that sample requirements have been greatly reduced. While higher protein concentrations are always better, significant qualitative information and some quantitative information (e.g., ligand screens) can be obtained from NMR at low µM concentration of unlabelled protein, and at concentrations above a few hundred µM, natural abundance heteronuclear NMR experiments are possible on 'unlabelled' samples that have been purified from any source.

The table below provides a guide to the types of information you can expect depending on the sample you have.

 

Sample concentration and labelling

What information from NMR can be obtained with that sample?

unlabelled protein at low µM

1D 1H spectra providing information on folding and the effects of solvent (pH, salt, etc.) and temperature, sample quality/reproducibility, ligand-detected small molecule screens (fast and quantitative), translational diffusion.

unlabelled protein at 500 µM to 1 mM

2D natural abundance 15N and 13C spectra to identify sample homogeneity, proportion of residues folded or flexible, some amino acid type specific information (e.g., glycines), greater information on sample quality and suitability for further studies; comparative studies become much more powerful – e.g., between different constructs (deletion or mutation) and different solvent conditions or with/without binding partners. 

15N or 13C labelled at 100-200 µM and above

Essentially any of the experiments described in this document can be performed. Some experiments are more demanding with regard to sample concentration, notably the CPMG experiments for quantifying µs-ms timescale motions.

Post-translationally labelled protein: protein expressed unlabelled and purified from any host, and then post-translationally labelled, typically by addition of 13C or 19F isotopes to lysines or cysteines.

Monitoring conformational changes upon ligand binding, for example to identify and utilise spectral signatures or to screen for ligand binding; quantifying dynamics.