Buffer optimization: Difference between revisions

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


#slow precipitation  
#slow precipitation  
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<br>  
<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>  
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.
 
<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.
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>  


==== '''Abbreviations:'''  ====
==== '''Abbreviations:'''  ====
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DTT: Dithiothreitol  
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  
MES: 2-(''N''-morpholino)ethanesulfonic acid  
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Tris: tris(hydroxymethyl)aminomethane;  
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  ====
==== <br> <br> References  ====


<references />
<references />

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:

  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.[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

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