Structure determination by NMR typically requires a protein concentration of 0.5 mM or greater, stable for several days at the desired temperature, usually 20-40^oC. Proteins and peptides should be dissolved in a suitable buffer (10-50 mM) with 10% D₂O. Ideally, we would aim for an acidic buffer (pH 4-7), but dissolve the sample in a buffer that provides maximum stability and solubility for your sample.

As the sample needs to be stable for days, the buffer content plays a critical role in protein/peptide sample stability. Buffer optimization may be used to improve sample stability and avoid the following issues:

• slow precipitation
• mixture of folded and unfolded protein
• aggregation problems
• degradation.

A screening with several buffers (with different pHs and containing chemicals such as detergents, protease inhibitor cocktails, arginine etc.) is therefore recommended to optimize NMR samples. These buffers usually contain salts (i.e. NaCl or KCl) which often increase protein solubility but may reduce signal to noise in cold NMR probes and so are worth screening also. Metal chelators (EDTA) and sodium azide at less than 50 μM should be used to inhibit microbial growth in biological samples where possible. If you have free thiols reducing agents, DTT or TCEP (1-5mM), may also be needed.