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 13 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 and optimization buffers:

MJ001        20mM MES, pH 6.5, 100mM NaCl, 5mM CaCl₂, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1x Proteinase Inhibitors, 5% D₂O

MJ002        20mM NH₄OAc, pH 5.5, 100mM NaCl, 5mM CaCl₂, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1X Proteinase Inhibitors, 5% D₂O

MJ003        20mM NH₄OAc, pH 4.5, 100mM NaCl, 5mM CaCl₂, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1X Proteinase Inhibitors, 5% D₂O

MJ004        50mM NH₄OAc, pH 5.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN₃, 1X Proteinase inhibitors, 5% D₂O

MJ005        50mM NH₄OAc, pH 5.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN₃, 1X Proteinase inhibitors, 5% D₂O

MJ006        50mM MES, pH 6.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN₃, 1X Proteinase inhibitors, 5% D₂O

MJ007        50mM MES, pH 6.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN₃, 1X Proteinase inhibitors, 5% D₂O

MJ008        25mM Na₂PO₄, pH 6.5, 450mM NaCl, 10mM DTT, 20uM ZnSO₄, 50 uM DSS, 0.02% NaN₃, 1X Proteinase inhibitors, 5% D₂O

MJ009        20mM MES, pH 6.5, 100mM NaCl, 5% Acetonitrile, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1X Proteinase Inhibitors, 5% D₂O

MJ010        20mM MES, pH 6.5, 100mM NaCl, 50mM Arginine, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1X Proteinase Inhibitors, 5% D₂O

MJ011        20mM MES, pH 6.5, 100mM NaCl, 1% Zwitter, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1X Proteinase Inhibitors, 5% D₂O

MJ012        20mM MES, pH 6.5, 100mM NaCl,  50uM ZnSO4, 10mM DTT, 50 uM DSS, 0.02% NaN₃, 1X Proteinase Inhibitors, 5% D₂O

Low Salt     10mM Tris-HCl, pH 7.5, 5mM DTT, 100mM NaCl , 0.02% NaN₃, 50 uM DSS, 1X Proteinase Inhibitors, 5% D₂O

Abbreviations:

DTT: Dithiothreitol

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

MES: 2-(N-morpholino)ethanesulfonic acid

Tris: tris(hydroxymethyl)aminomethane;

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

DSS: 2,2-Dimethyl-2-silapentane-5-sulfonic acid