Buffer optimization: Difference between revisions

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Buffer optimization may be used to improve a protein sample with:<o:p></o:p>
Buffer optimization may be used to improve a protein sample with:


#
slow precipitation<o:p></o:p>


#
mixture of folded and unfolded protein<o:p></o:p>


#
slow precipitation
aggregation problems<o:p></o:p>


#
mixture of folded and unfolded protein
multiple populations (too many peaks) for other reasons<o:p></o:p>


#
aggregation problems
and many other reasons<o:p></o:p>


<!--[if !supportEmptyParas]-->&nbsp;<!--[endif]--><o:p></o:p>
multiple populations (too many peaks) for 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.<o:p></o:p>
and many other reasons&lt;o:p&gt;&lt;/o:p&gt;


<br>


==== '''NMR screening buffer:<o:p></o:p>''' ====


pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole
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.
<br>


pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x
==== '''NMR screening buffer:'''  ====


pH 5.5, 20 mM NH4OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x
pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole


pH 4.5, 20mM NH4OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x
pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x  


pH 5.0, 50 mM NH4OAc, 50 mM Arginine, protease inhibitor 1x
pH 5.5, 20 mM NH4OAc, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, protease inhibitor 1x  


pH 5.0, 50 mM NH4OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x
pH 4.5, 20mM NH4OAc, 100 mM NaCl, 5mM CaCl<sub>2</sub>, protease inhibitor 1x  


pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x
pH 5.0, 50 mM NH4OAc, 50 mM Arginine, protease inhibitor 1x  


pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x
pH 5.0, 50 mM NH4OAc, 5% CH<sub>3</sub>CN, protease inhibitor 1x  


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


pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x
pH 6.0, 50 mM MES, 5% CH<sub>3</sub>CN, protease inhibitor 1x  


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


pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter
pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH<sub>3</sub>CN, protease inhibitor 1x


pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x  


<!--[if !supportEmptyParas]-->&nbsp;<!--[endif]--><o:p></o:p>
pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter


All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O.<o:p></o:p>
pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO<sub>4</sub>, protease inhibitor 1x


<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.<o:p></o:p>


<br>
All buffers contain 0.02% NaN<sub>3</sub>, 10 mM DTT (or 1 mM TCEP), and 5% D<sub>2</sub>O.


==== '''Abbreviations:<o:p></o:p>''' ====
<br>  


DTT: Dithiothreitol<o:p></o:p>
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.


Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.<span style="">&nbsp; </span>963015 (CALBIOCHEM)<o:p></o:p>
<br>  


MES: 2-(''N''<span style="font-style: normal;">-morpholino)ethanesulfonic
==== '''Abbreviations:''' ====
acid<o:p></o:p></span>


Tris: tris(hydroxymethyl)aminomethane<o:p></o:p>
DTT: Dithiothreitol


Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE).<o:p></o:p>
Zwitter: ZWITTERAGENT<sup>®</sup> 3-12 Detergent cat.963015 (CALBIOCHEM)


TCEP: ''tris''<span style="font-style: normal;">(2-carboxyethyl)phosphine<o:p></o:p></span>
MES: 2-(''N''-morpholino)ethanesulfonic
acid


'''<!--[if !supportEmptyParas]-->&nbsp;<!--[endif]--><o:p></o:p>'''
Tris: tris(hydroxymethyl)aminomethane;  


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


<br>
TCEP: ''tris''(2-carboxyethyl)phosphine
 
 
<br> <br>

Revision as of 16:17, 19 November 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<o:p></o:p>


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 buffer:

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




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