Buffer optimization

Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues:

1. slow precipitation
2. mixture of folded and unfolded protein
3. aggregation problems
4. multiple populations (too many peaks) for other reasons
5. and many other reasons

At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by ¹H-¹⁵N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.

NMR screening buffers:

pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole

pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl₂, protease inhibitor 1x

pH 5.5, 20 mM NH₄OAc, 100 mM NaCl, 5 mM CaCl₂, protease inhibitor 1x

pH 4.5, 20mM NH₄OAc, 100 mM NaCl, 5mM CaCl₂, protease inhibitor 1x

pH 5.0, 50 mM NH₄OAc, 50 mM Arginine, protease inhibitor 1x

pH 5.0, 50 mM NH₄OAc, 5% CH₃CN, protease inhibitor 1x

pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x

pH 6.0, 50 mM MES, 5% CH₃CN, protease inhibitor 1x

pH 6.5, 25 mM Na₂PO₄, 450 mM NaCl, 20 mM ZnSO₄, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH₃CN, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter

pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO₄, protease inhibitor 1x

All buffers contain 0.02% NaN₃, 10 mM DTT (or 1 mM TCEP), and 5% D₂O.

TCEP is used instead of DTT, when the protein is eluted from the Ni-column.  Otherwise, Ni⁺² and DTT form an insoluble brown precipitate.

Abbreviations:

DTT: Dithiothreitol

Zwitter: ZWITTERAGENT^® 3-12 Detergent cat.963015 (CALBIOCHEM)

MES: 2-(N-morpholino)ethanesulfonic acid

Tris: tris(hydroxymethyl)aminomethane;

Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE);

TCEP: tris(2-carboxyethyl)phosphine