NMR Buffer Conditions Optimization

  1. 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.
  2. 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.
  3. ~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.)
  4. ~ 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.


  1. 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.
  2. 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