Buffer optimization - Revision history

Krishna at 23:01, 26 November 2012

2012-11-26T23:01:28Z

← Older revision		Revision as of 23:01, 26 November 2012
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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

Jma at 17:21, 5 October 2011

2011-10-05T17:21:39Z

← Older revision		Revision as of 17:21, 5 October 2011
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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 <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>		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 and optimization buffers:''' ====		==== '''NMR screening and optimization buffers:''' ====

	MJ001        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		MJ001        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

			MJ002        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>

	~~MJ002~~        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>		MJ003        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>

	~~MJ003~~        ~~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>~~		MJ004        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

	~~MJ004~~        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		MJ005        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

	~~MJ005~~        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		MJ006        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

	~~MJ006~~        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		MJ007        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

	~~MJ007~~        ~~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		MJ008        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

	~~MJ008~~        ~~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		MJ009        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

	~~MJ009~~        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		MJ010        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

	~~MJ010 ~~       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		MJ011        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

	~~MJ011~~        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		MJ012        20mM MES, pH 6.5, 100mM NaCl,  50uM ZnSO4, 10mM DTT, 50 uM DSS, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O

	~~MJ012~~    ~~    20mM MES~~, pH 6.5, 100mM NaCl~~,  50uM ZnSO4, 10mM DTT, 50 uM DSS~~, 0.02% NaN<sub>3</sub>, 1X Proteinase Inhibitors, 5% D<sub>2</sub>O		Low Salt     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>

	~~Low Salt     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>~~
	~~<br>~~
	==== '''Abbreviations:''' ====		==== '''Abbreviations:''' ====

Jma at 17:20, 5 October 2011

2011-10-05T17:20:57Z

← Older revision		Revision as of 17:20, 5 October 2011
Line 9:		Line 9:
	<br>		<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. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</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:''' ====		==== '''NMR screening and optimization buffers:''' ====

	pH 7.5, 50 ~~mM Tris~~, ~~500 mM NaCl~~, ~~500 mM Imidazole~~		MJ001        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 6.5, ~~20 mM MES, 100 mM~~ NaCl, ~~5 mM~~ CaCl<sub>2</sub>, ~~protease inhibitor 1x~~		MJ002        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 5.5, 20 mM~~ NH<sub>4</sub>OAc, ~~100 mM~~ NaCl, ~~5 mM~~ CaCl<sub>2</sub>, ~~protease inhibitor 1x~~		MJ003        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 4.5, ~~20mM NH~~<sub>4</sub>~~OAc~~, ~~100 mM NaCl~~, ~~5mM CaCl~~<sub>2</sub>~~, protease inhibitor 1x~~		MJ004        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 5.0, 50 ~~mM NH~~<sub>4</sub>~~OAc~~, ~~50 mM Arginine~~, ~~protease inhibitor 1x~~		MJ005        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 5.0, 50 ~~mM NH~~<sub>4</sub>~~OAc~~, 5% CH<sub>3</sub>~~CN, protease inhibitor 1x~~		MJ006        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 6.0, 50 ~~mM MES~~, ~~50 mM Arginine~~, ~~protease inhibitor 1x~~		MJ007        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 6.0, 50 ~~mM MES~~, 5% CH<sub>3</sub>CN, ~~protease inhibitor 1x~~		MJ008        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, ~~25 mM Na<sub>2</sub>PO~~<sub>4</sub>, ~~450 mM NaCl~~, ~~20 mM ZnSO~~<sub>4</sub>~~, protease inhibitor 1x~~		MJ009        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~~, 5% CH<sub>3</sub>CN, ~~protease inhibitor 1x~~		MJ010        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 Arginine~~, ~~protease inhibitor 1x~~		MJ011        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

	pH 6.5, ~~20 mM MES~~, ~~100 mM NaCl~~, 1% ~~Zwitter~~		MJ012        20mM MES, pH 6.5, 100mM NaCl,  50uM ZnSO4, 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~~		Low Salt     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>
	~~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="">  </span>Otherwise, Ni<sup>+2</sup> and DTT form an insoluble brown precipitate.~~

	<br>

	==== '''Abbreviations:''' ====		==== '''Abbreviations:''' ====

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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 />

Jma at 16:23, 5 October 2011

2011-10-05T16:23:04Z

← Older revision		Revision as of 16:23, 5 October 2011
Line 65:		Line 65:
	==== <br> <br> References ====		==== <br> <br> References ====

	[<references />]		<references />

Jma at 16:22, 5 October 2011

2011-10-05T16:22:32Z

← Older revision		Revision as of 16:22, 5 October 2011
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	==== <br> <br> References ====		==== <br> <br> References ====

	[~~http://www.ncbi.nlm.nih.gov/pubmed/19915800~~ <references />]		[<references />]

Jma at 16:21, 5 October 2011

2011-10-05T16:21:01Z

← Older revision		Revision as of 16:21, 5 October 2011
Line 65:		Line 65:
	==== <br> <br> References ====		==== <br> <br> References ====

	<references />		[http://www.ncbi.nlm.nih.gov/pubmed/19915800 <references />]

Jma at 16:20, 5 October 2011

2011-10-05T16:20:14Z

← Older revision		Revision as of 16:20, 5 October 2011
Line 9:		Line 9:
	<br>		<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. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', ~~'''~~46, 11-22.</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 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. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', 46, 11-22.</ref><br>

	==== '''NMR screening buffers:''' ====		==== '''NMR screening buffers:''' ====

Jma at 16:19, 5 October 2011

2011-10-05T16:19:26Z

← Older revision		Revision as of 16:19, 5 October 2011
Line 9:		Line 9:
	<br>		<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 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. (2010). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''46, 11-22.</ref><br>

	==== '''NMR screening buffers:''' ====		==== '''NMR screening buffers:''' ====

Jma at 16:18, 5 October 2011

2011-10-05T16:18:11Z

← Older revision		Revision as of 16:18, 5 October 2011
Line 65:		Line 65:
	==== <br> <br> References ====		==== <br> <br> References ====

	~~[http://www.ncbi.nlm.nih.gov/pubmed/19915800 ]~~<references />		<references />

Jma at 16:12, 5 October 2011

2011-10-05T16:12:45Z

← Older revision		Revision as of 16:12, 5 October 2011
Line 65:		Line 65:
	==== <br> <br> References ====		==== <br> <br> References ====

	[http://www.ncbi.nlm.nih.gov/pubmed/19915800 1. 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.  A microscale protein NMR sample screening pipeline.  (2010)  ''J. Biomol. NMR 46'', 11-22.]~~ ~~		[http://www.ncbi.nlm.nih.gov/pubmed/19915800 ]<references />