Buffer optimization: Difference between revisions
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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.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> | 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.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref><br> | ||
==== '''NMR screening | ==== '''NMR screening buffers:''' ==== | ||
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole | pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole | ||
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TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style=""> </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. | TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style=""> </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate. | ||
<br> | <br> | ||
==== '''Abbreviations:''' ==== | ==== '''Abbreviations:''' ==== | ||
Revision as of 20:26, 3 December 2009
Buffer optimization may be used to improve a protein sample with:
- slow precipitation
- mixture of folded and unfolded protein
- aggregation problems
- multiple populations (too many peaks) for other reasons
- 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.[1]
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
References
- ↑ Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." J. Biomol. NMR, in press.