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Although the MCSB 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, of solution biomolecular NMR experiments. For more information on sample preparation
of membrane protein studies with either solution or solid-state NMR techniques please contact our expertise and refer to relevant
references.
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 550-600 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.
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