- For homonuclear NMR expt., Na/K phosphate (pH5.0 to 7.5), Tris-d11 (pH7.5 to 8.5), or non-proton containing compounds can be used as the buffer; for heteronuclear NMR expt., protonated buffers are fine.
- In general, around physiological pH (6-7) and salt (100-150 mM) conditions are the best, low pH (< 6) can give good quality spectra due to slow NH exchange, but the protein structures could be influenced.
- ~5mM DTT, ~5mM EDTA, and ~0.02% NaN3 are commonly included in the buffer. (In addition, ~10mM CHAPS, a non-denaturing detergent, may prevent the protein aggregation caused by hydrophobic interaction (J. Biomol. NMR, 1993, 3, 121-126.), and Glycine (molar scaled) may stabilize the protein and prevent from heat-denaturing.)
- ~ 0.05mM of the N15 labeled protein sample is needed for 2D N15 HSQC; and ~ 0.35mM of the C13/N15 labeled one is good for 3D triple-resonance expts. for the backbone assignments, and on 600 or 800 MHz spectrometers equipped with cryoprobes.
Note:
- Do literature search first for your protein’s e.g. x-ray structure paper or some proteins’ in the same family of your protein, and use their pH and salt concentration as your starting NMR buffer condition.
- Use other methods, e.g. analytical ultra-centrifugation or dynamic light scattering to screen pH and salt concentration, and experimentally prove that your protein is mono-disperse state (either uniformly a monomer or dimer, trimer, tetramer, etc.) under the chosen NMR buffer condition, but not a multi-disperse mixture.
Hongwei edited on 9/1/2023