NMR Facility - Sample Requirement
Although the NCBS NMR facility is dedicated to all kinds of possible applications, biomolecular research is the major emphasis. Biomacromolecular solution NMR study is the most important focus. Listed below are some basic common requirements for sample preparation, in particular protein samples in solution biomolecular NMR experiments.
- Common Profile: 0.1-2.5 mM concentration, typically about 1 mM; stable at room temperature over weeks; 2H/13C/15N isotope labeling; mono-disperse; purity greater than 95%; neither paramagnetic impurities nor solid particles present; organic buffer components in perdeuterated forms; 10-50 µM DSS or TSP as inner reference; 90%-95%H2O:10%-5%D2O.
- Volume: 500-550 µL for regular 5 mm NMR tubes and 260-300 µL for Shigemi tubes; 2-30 mg of protein of 8-30 kDa molecular weight, typically about 10 mg.
- Stability: No precipitation in proper buffers; no degradation with removal of proteases or addition of protease inhibitors; no decomposition by bacteria with addition of 10-50 µM NaN3 or use of micro-filtration; no oxidation (SH-groups) through addition of DTT.
- Buffer: pH: neutral or slightly acidic; phosphate buffer preferred; away from protein pI value. Ionic strength: less than 500 mM for conventional probes and less than 100 mM for cryogenic probes.
- Overexpression: E. coli strains preferred; chemically defined media (e.g. M9 and modifications) with 13C isotope enriched glucose/glycerol and 15N isotope enriched NH4Cl/(NH4)2SO4 as sole carbon source and nitrogen source, respectively, for uniformly 13C/15N labeling; protein yield more than 5 mg/L preferred.