Knight:Purification of His-tagged proteins/Native - Revision history

Reshma P. Shetty at 22:36, 5 December 2006

Reshma P. Shetty — Tue, 05 Dec 2006 22:36:41 GMT

←Older revision		Revision as of 22:36, 5 December 2006
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	#PierceCellLysis [http://www.piercenet.com/Proteomics/browse.cfm?fldID=72F377CD-2581-438C-9B27-5360226EA128 Cell lysis technical information from Pierce]		#PierceCellLysis [http://www.piercenet.com/Proteomics/browse.cfm?fldID=72F377CD-2581-438C-9B27-5360226EA128 Cell lysis technical information from Pierce]
	</biblio>		</biblio>
		+
		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:Protein]]

Reshma P. Shetty: /* Notes */

Reshma P. Shetty — Mon, 27 Nov 2006 21:57:22 GMT

Notes

←Older revision		Revision as of 21:57, 27 November 2006
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	*Sauer lab uses a Qiagen Ni-NTA resin but this protocol uses spin columns. (Smaller scale purification).		*Sauer lab uses a Qiagen Ni-NTA resin but this protocol uses spin columns. (Smaller scale purification).
	*Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.		*Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.
-	*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column.	+	*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column. However, consider using a SlyD knockout strain. SlyD is a 20-25 kDa protein that has several histidines near each other and can often contaminate Ni column purifications from ''Escherichia coli''.
	*20 year old spin columns don't work. :)		*20 year old spin columns don't work. :)
	*The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins.		*The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins.
		+	*Can lyse cells by doing repeated freeze-thaw cycles at -80°C or sonication also works.

	==Safety==		==Safety==

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Thu, 05 Oct 2006 15:37:21 GMT

Procedure

←Older revision		Revision as of 15:37, 5 October 2006
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	#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''		#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''
	#*''It may be that the thaw process needs to take place at 37°C rather than in slushy ice.''<cite>PierceCellLysis</cite>		#*''It may be that the thaw process needs to take place at 37°C rather than in slushy ice.''<cite>PierceCellLysis</cite>
		+	#*''An alternative option is to use the bead beater to mechanically lyse the cells.''
	#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.		#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.
	#*''20 mins may be enough.''		#*''20 mins may be enough.''

Reshma P. Shetty at 15:35, 5 October 2006

Reshma P. Shetty — Thu, 05 Oct 2006 15:35:48 GMT

←Older revision		Revision as of 15:35, 5 October 2006
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	#Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.		#Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
	#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).		#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
-	#*''Maybe try 100mL cultures.''	+	#*''Maybe try 100mL cultures. 100mL was too much. I think 50mL might be preferable.''
	#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins at 4°C.		#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins at 4°C.
	#Decant supernatant.		#Decant supernatant.

Reshma P. Shetty at 22:49, 28 September 2006

Reshma P. Shetty — Thu, 28 Sep 2006 22:49:12 GMT

←Older revision		Revision as of 22:49, 28 September 2006
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	#*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''		#*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''
	#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''		#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''
		+	#*''It may be that the thaw process needs to take place at 37°C rather than in slushy ice.''<cite>PierceCellLysis</cite>
	#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.		#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.
	#*''20 mins may be enough.''		#*''20 mins may be enough.''
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	<biblio>		<biblio>
	#QiagenNTAManual [http://www1.qiagen.com/literature/handbooks/PDF/Protein/Purification/NiNTA_Spin/1023679HBNINTA_022003WW.pdf Qiagen Ni NTA Spin Kit manual]		#QiagenNTAManual [http://www1.qiagen.com/literature/handbooks/PDF/Protein/Purification/NiNTA_Spin/1023679HBNINTA_022003WW.pdf Qiagen Ni NTA Spin Kit manual]
		+	#PierceCellLysis [http://www.piercenet.com/Proteomics/browse.cfm?fldID=72F377CD-2581-438C-9B27-5360226EA128 Cell lysis technical information from Pierce]
	</biblio>		</biblio>

Reshma P. Shetty at 19:52, 28 September 2006

Reshma P. Shetty — Thu, 28 Sep 2006 19:52:47 GMT

←Older revision		Revision as of 19:52, 28 September 2006
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	#Incubate cells on ice for 30 mins.		#Incubate cells on ice for 30 mins.
	#Freeze the cells at -80°C and thaw for 3 cycles.		#Freeze the cells at -80°C and thaw for 3 cycles.
		+	#*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''
	#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''		#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''
	#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.		#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Thu, 28 Sep 2006 19:39:57 GMT

Procedure

←Older revision		Revision as of 19:39, 28 September 2006
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	#Add 10 μL 100 mg/mL lysozyme to 1 mg/mL final concentration.		#Add 10 μL 100 mg/mL lysozyme to 1 mg/mL final concentration.
	#Add a protease inhibitor here?		#Add a protease inhibitor here?
		+	#*''Trying 1/4 of a mini, EDTA-free protease inhibitor cocktail tablet from Roche.''
	#Incubate cells on ice for 30 mins.		#Incubate cells on ice for 30 mins.
-	#~~Sonicate or homogenize on ice to lyse~~ cells.	+	#Freeze the cells at -80°C and thaw for 3 cycles.
-	#*''~~Six~~ times for 10s each time with 5s pauses in between.''	+	#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''
-	#Centrifuge lysate at 10000 x ''g'' for ~~20-~~30 mins at 4°C to pellet cellular debris. Collect supernatant.	+	#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4°C to pellet cellular debris. Collect supernatant.
		+	#*''20 mins may be enough.''
	#Add 600 μL lysis buffer to Ni-NTA column to equilibrate.		#Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid to remove equilibration buffer at 4°C.		#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid to remove equilibration buffer at 4°C.

Reshma P. Shetty at 16:21, 28 September 2006

Reshma P. Shetty — Thu, 28 Sep 2006 16:21:22 GMT

←Older revision		Revision as of 16:21, 28 September 2006
Line 79:		Line 79:
	*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column.		*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column.
	*20 year old spin columns don't work. :)		*20 year old spin columns don't work. :)
		+	*The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins.

	==Safety==		==Safety==

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Tue, 26 Sep 2006 01:43:42 GMT

Procedure

←Older revision		Revision as of 01:43, 26 September 2006
Line 32:		Line 32:
	#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).		#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
	#*''Maybe try 100mL cultures.''		#*''Maybe try 100mL cultures.''
-	#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins.	+	#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins at 4°C.
-	~~#*''The Qiagen protocol didn't specify a temperature so I did~~ 4°C.''	+
	#Decant supernatant.		#Decant supernatant.
	#The cell pellet can be stored at -70°C or processed immediately.		#The cell pellet can be stored at -70°C or processed immediately.
-	#*''I stored the ~~pellet at -80°C~~.''	+	#Chill the following on ice ...
		+	#*lysis buffer
		+	#*wash buffer
		+	#*elution buffer
		+	#*2mL eppendorf tube
	#Thaw for 15 mins on ice.		#Thaw for 15 mins on ice.
	#*''May take longer than 15 mins.''		#*''May take longer than 15 mins.''
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	#Resuspend in 1mL lysis buffer (see above).		#Resuspend in 1mL lysis buffer (see above).
	#*''The lysis buffer contains 10mM imidazole to reduce untagged, contaminating proteins. The imidazole concentration can be reduced to either 1-5 mM or increased to 20 mM as appropriate.''		#*''The lysis buffer contains 10mM imidazole to reduce untagged, contaminating proteins. The imidazole concentration can be reduced to either 1-5 mM or increased to 20 mM as appropriate.''
-	#Add 10 μ~~/mL~~ 100 mg/mL lysozyme to 1 mg/mL final concentration.	+	#Add 10 μL 100 mg/mL lysozyme to 1 mg/mL final concentration.
		+	#Add a protease inhibitor here?
	#Incubate cells on ice for 30 mins.		#Incubate cells on ice for 30 mins.
	#Sonicate or homogenize on ice to lyse cells.		#Sonicate or homogenize on ice to lyse cells.
Line 48:		Line 52:
	#Centrifuge lysate at 10000 x ''g'' for 20-30 mins at 4°C to pellet cellular debris. Collect supernatant.		#Centrifuge lysate at 10000 x ''g'' for 20-30 mins at 4°C to pellet cellular debris. Collect supernatant.
	#Add 600 μL lysis buffer to Ni-NTA column to equilibrate.		#Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
-	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid to remove equilibration buffer.	+	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid to remove equilibration buffer at 4°C.
	#Save 20μL cleared lysate.		#Save 20μL cleared lysate.
	#Load 600 μL cleared lysate to Ni-NTA column.		#Load 600 μL cleared lysate to Ni-NTA column.
-	#Centrifuge Ni-NTA column 5 mins at 700 x ''g'' with closed lid.	+	#Centrifuge Ni-NTA column 5 mins at 700 x ''g'' with closed lid at 4°C.
	#*''The closed lid increases binding time.''		#*''The closed lid increases binding time.''
	#*''Repeat this step to load the rest of my cleared lysate?''		#*''Repeat this step to load the rest of my cleared lysate?''
	#*''Save flow through.''		#*''Save flow through.''
	#Add 600 μL wash buffer to Ni-NTA column.		#Add 600 μL wash buffer to Ni-NTA column.
-	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.	+	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
	#*''Save flow through.''		#*''Save flow through.''
	#Add 600 μL wash buffer to Ni-NTA column.		#Add 600 μL wash buffer to Ni-NTA column.
-	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.	+	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
	#*''Save flow through.''		#*''Save flow through.''
	#Tranfer to clean 1.5mL eppendorf tube.		#Tranfer to clean 1.5mL eppendorf tube.
	#Add 200μL elution buffer.		#Add 200μL elution buffer.
-	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.	+	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
	#*''Most of the protein should elute in this elution step.''		#*''Most of the protein should elute in this elution step.''
	#Tranfer to clean 1.5mL eppendorf tube.		#Tranfer to clean 1.5mL eppendorf tube.
	#Add 200μL elution buffer.		#Add 200μL elution buffer.
-	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.	+	#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
	#*''Just in case.''		#*''Just in case.''

Reshma P. Shetty at 20:25, 25 September 2006

Reshma P. Shetty — Mon, 25 Sep 2006 20:25:53 GMT

←Older revision		Revision as of 20:25, 25 September 2006
Line 42:		Line 42:
	#Resuspend in 1mL lysis buffer (see above).		#Resuspend in 1mL lysis buffer (see above).
	#*''The lysis buffer contains 10mM imidazole to reduce untagged, contaminating proteins. The imidazole concentration can be reduced to either 1-5 mM or increased to 20 mM as appropriate.''		#*''The lysis buffer contains 10mM imidazole to reduce untagged, contaminating proteins. The imidazole concentration can be reduced to either 1-5 mM or increased to 20 mM as appropriate.''
-	#Add lysozyme to 1 mg/mL concentration.	+	#Add 10 μ/mL 100 mg/mL lysozyme to 1 mg/mL final concentration.
	#Incubate cells on ice for 30 mins.		#Incubate cells on ice for 30 mins.
	#Sonicate or homogenize on ice to lyse cells.		#Sonicate or homogenize on ice to lyse cells.