Buffer optimization: Difference between revisions

Latest revision as of 23:01, 26 November 2012

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

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 13 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 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.693015](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

References

↑ Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." J. Biomol. NMR, 46, 11-22.

[1] Rossi, P. et. al. (2010). "A microscale protein NMR sample screening pipeline." J. Biomol. NMR, 46, 11-22.

[1]

@@ Line 9: / Line 9: @@
 <br>
-At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by <sup>1</sup>H-<sup>15</sup>N 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 <sup>1</sup>H-<sup>15</sup>N 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.<ref>Rossi, P. et. al. (2010).  "A microscale protein NMR sample screening pipeline."  ''J. Biomol. NMR'', 46, 11-22.</ref><br>
-==== '''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 CaCl<sub>2</sub>, protease inhibitor 1x
+==== '''NMR screening and optimization buffers:'''  ====
-pH 5.5, 20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x
+MJ001&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1x Proteinase Inhibitors, 5% D<sub>2</sub>O
-pH 4.5, 20mM NH<sub>4</sub>OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x
+MJ002&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM NH<sub>4</sub>OAc, pH 5.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br>
-pH 5.0, 50 mM NH<sub>4</sub>OAc, 50 mM Arginine, protease inhibitor 1x
+MJ003&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM NH<sub>4</sub>OAc, pH 4.5, 100mM NaCl, 5mM CaCl<sub>2</sub>, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br>
-pH 5.0, 50 mM NH<sub>4</sub>OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x
+MJ004&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM NH<sub>4</sub>OAc, pH 5.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O
-pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x
+MJ005&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM NH<sub>4</sub>OAc, pH 5.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O
-pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x
+MJ006&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM MES, pH 6.0, 10mM DTT, 50mM Arginine, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O
-pH 6.5, 25 mM Na<sub>2</sub>PO<sub>4</sub>, 450 mM NaCl, 20 mM ZnSO<sub>4</sub>, protease inhibitor 1x
+MJ007&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50mM MES, pH 6.0, 10mM DTT, 5% Acetonitrile, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O
-pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x
+MJ008&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 25mM Na<sub>2</sub>PO<sub>4</sub>, pH 6.5, 450mM NaCl, 10mM DTT, 20uM ZnSO<sub>4</sub>, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase inhibitors, 5% D<sub>2</sub>O
-pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x
+MJ009&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 5% Acetonitrile, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O
-pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter
+MJ010&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl, 50mM Arginine, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O
-pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x
+MJ011&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; 20mM MES, pH 6.5, 100mM NaCl, 1% Zwitter, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O
-<br>
+MJ012&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20mM MES, pH 6.5, 100mM NaCl,&nbsp; 50uM ZnSO4, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O
-All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O.
+Low Salt&nbsp;&nbsp;&nbsp;&nbsp; 10mM Tris-HCl, pH 7.5, 5mM DTT, 100mM NaCl , 0.02% NaN<sub>3</sub>, 50 uM DSS, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O<br>
-<br>
+<br> <br>
-TCEP is used instead of DTT, when the protein is eluted from the Ni-column.<span style="">&nbsp; </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate.
-<br>
 ==== '''Abbreviations:'''  ====
@@ Line 53: / Line 47: @@
 DTT: Dithiothreitol
-Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM)
+Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent [cat.693015](CALBIOCHEM)
 MES: 2-(''N''-morpholino)ethanesulfonic acid
@@ Line 59: / Line 53: @@
 Tris: tris(hydroxymethyl)aminomethane;
-Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE);
+Protease inhibitors: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE);
-TCEP: ''tris''(2-carboxyethyl)phosphine
+DSS: 2,2-Dimethyl-2-silapentane-5-sulfonic acid
 ==== <br> <br> References  ====
-. Rossi, P. Swapna, G.V.T., Huang, Y.J., Aramini, J.M., Anklin, C., Conover, K., Hamilton, K., Xiao, R., Acton, T.B., Ertekin, A., Everett, J.K., and Montelione, G.T.&nbsp; A microscale protein NMR sample screening pipeline.&nbsp; (2010)&nbsp; ''J. Biomol. NMR 46'', 11-22.&nbsp;
+<references />

Buffer optimization: Difference between revisions

Latest revision as of 23:01, 26 November 2012

NMR screening and optimization buffers:

Abbreviations:

References

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