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

Reshma P. Shetty: Reverted edits by Felipe Leprevost (Talk); changed back to last version by Reshma P. Shetty

Reshma P. Shetty — Wed, 10 Oct 2007 14:04:49 GMT

Reverted edits by Felipe Leprevost (Talk); changed back to last version by Reshma P. Shetty

←Older revision		Revision as of 14:04, 10 October 2007
Line 16:		Line 16:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*20 mM imidazole (~~adjusted~~ by ~~Leprevost~~, 10/03/~~2007~~)	+	*10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
	*pH 6.3		*pH 6.3
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)
Line 24:		Line 24:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*~~250~~ mM imidazole (~~adjusted~~ by ~~Leprevost~~, 10/03/~~2007~~)	+	*10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
	*pH 4.5		*pH 4.5
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)
Line 31:		Line 31:
	*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume)		*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume)
	*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well?		*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well?
-	*Leprevost adjusted the Imidazol concentration in Washing and Elution buffers. A high Imidazol concentration is needed in order to elute the 6xHis-tagged protein.
	*The urea should be freshly prepared and deionized prior to use.		*The urea should be freshly prepared and deionized prior to use.
	*The buffers should each be degassed before use.		*The buffers should each be degassed before use.

Felipe Leprevost: /* Wash buffer (Qiagen buffer C) */

Felipe Leprevost — Wed, 03 Oct 2007 16:11:08 GMT

Wash buffer (Qiagen buffer C)

←Older revision		Revision as of 16:11, 3 October 2007
Line 16:		Line 16:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*20 mM imidazole (adjusted by Leprevost, 10/3/2007)	+	*20 mM imidazole (adjusted by Leprevost, 10/03/2007)
	*pH 6.3		*pH 6.3
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)

Felipe Leprevost: /* Elution buffer (Qiagen buffer E) */

Felipe Leprevost — Wed, 03 Oct 2007 16:10:56 GMT

Elution buffer (Qiagen buffer E)

←Older revision		Revision as of 16:10, 3 October 2007
Line 24:		Line 24:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*250 mM imidazole	+	*250 mM imidazole (adjusted by Leprevost, 10/03/2007)
	*pH 4.5		*pH 4.5
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)

Felipe Leprevost: /* Wash buffer (Qiagen buffer C) */

Felipe Leprevost — Wed, 03 Oct 2007 16:10:24 GMT

Wash buffer (Qiagen buffer C)

←Older revision		Revision as of 16:10, 3 October 2007
Line 16:		Line 16:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*20 mM imidazole	+	*20 mM imidazole (adjusted by Leprevost, 10/3/2007)
	*pH 6.3		*pH 6.3
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)

Felipe Leprevost: /* Notes */

Felipe Leprevost — Wed, 03 Oct 2007 16:08:56 GMT

Notes

←Older revision		Revision as of 16:08, 3 October 2007
Line 31:		Line 31:
	*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume)		*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume)
	*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well?		*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well?
		+	*Leprevost adjusted the Imidazol concentration in Washing and Elution buffers. A high Imidazol concentration is needed in order to elute the 6xHis-tagged protein.
	*The urea should be freshly prepared and deionized prior to use.		*The urea should be freshly prepared and deionized prior to use.
	*The buffers should each be degassed before use.		*The buffers should each be degassed before use.

Felipe Leprevost: /* Elution buffer (Qiagen buffer E) */

Felipe Leprevost — Wed, 03 Oct 2007 16:05:30 GMT

Elution buffer (Qiagen buffer E)

←Older revision		Revision as of 16:05, 3 October 2007
Line 24:		Line 24:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*10 mM imidazole ~~(recommended by Kathleen, 9/27/2006, also use here?)~~	+	*250 mM imidazole
	*pH 4.5		*pH 4.5
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)

Felipe Leprevost: /* Wash buffer (Qiagen buffer C) */

Felipe Leprevost — Wed, 03 Oct 2007 16:05:05 GMT

Wash buffer (Qiagen buffer C)

←Older revision		Revision as of 16:05, 3 October 2007
Line 16:		Line 16:
	*100 mM NaH<sub>2</sub>PO<sub>4</sub>		*100 mM NaH<sub>2</sub>PO<sub>4</sub>
	*10 mM Tris Cl		*10 mM Tris Cl
-	*10 mM imidazole ~~(recommended by Kathleen, 9/27/2006, also use here?)~~	+	*20 mM imidazole
	*pH 6.3		*pH 6.3
	*1 mM TCEP (add just before use)		*1 mM TCEP (add just before use)

Reshma P. Shetty at 16:36, 16 January 2007

Reshma P. Shetty — Tue, 16 Jan 2007 16:36:30 GMT

←Older revision		Revision as of 16:36, 16 January 2007
Line 45:		Line 45:
	#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.''	+	#*''I stored the pellet at -80°C. Long term storage at -80°C might affect protein recovery???''

	===Prepare solutions===		===Prepare solutions===
Line 51:		Line 51:
	#[[Knight:Deionization\|Deionize]] the urea solution.		#[[Knight:Deionization\|Deionize]] the urea solution.
	#Prepare lysis, wash and elution buffers (everything except reducing agent).		#Prepare lysis, wash and elution buffers (everything except reducing agent).
-	~~#Degas lysis, wash and elution buffers.~~
-	~~#*Place under vacuum for one hour.~~
-	~~#Add TCEP to 1mM final concentration.~~
	#[[Knight:pH meter/Measurement\|Verify pH]] of lysis, wash and elution buffers. Adjust if necessary.		#[[Knight:pH meter/Measurement\|Verify pH]] of lysis, wash and elution buffers. Adjust if necessary.
	#*''Dissociation of urea can lead to changes in pH. The pH definitely needs to be checked prior to using the solutions.''		#*''Dissociation of urea can lead to changes in pH. The pH definitely needs to be checked prior to using the solutions.''
		+	#Degas lysis, wash and elution buffers by placing under vacuum for one hour.
		+	#*''Tom recommends doing the degassing step last.''
		+	#Add TCEP to 1mM final concentration.
		+	#*''TCEP is not stable in phosphate buffers at neutral pH.''
		+	#Verify that pH hasn't changed.
		+	#*''It is not known whether TCEP affects pH. Degassing can affect pH in some cases.''

	===Purify protein===		===Purify protein===

Reshma P. Shetty: /* Wash buffer (Qiagen buffer C) */

Reshma P. Shetty — Mon, 15 Jan 2007 16:30:21 GMT

Wash buffer (Qiagen buffer C)

←Older revision		Revision as of 16:30, 15 January 2007
Line 18:		Line 18:
	*10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)		*10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
	*pH 6.3		*pH 6.3
-	*10 mM TCEP (add just before use)	+	*1 mM TCEP (add just before use)

	===Elution buffer (Qiagen buffer E)===		===Elution buffer (Qiagen buffer E)===

Reshma P. Shetty: /* Purify protein */

Reshma P. Shetty — Mon, 18 Dec 2006 14:26:09 GMT

Purify protein

←Older revision		Revision as of 14:26, 18 December 2006
Line 63:		Line 63:
	#Transferred to 2mL eppendorf tube.		#Transferred to 2mL eppendorf tube.
	#Resuspend in 1mL Lysis Buffer (see above).		#Resuspend in 1mL Lysis Buffer (see above).
		+	#Add half or 1/4 protease inhibitor pellet.
	#Incubate cells with agitation for 1 hr at room temperature.		#Incubate cells with agitation for 1 hr at room temperature.
	#*''Use an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.''		#*''Use an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.''
-	~~#Add half or 1/4 protease inhibitor pellet.~~
	#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.		#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
	#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.
	#Save 20 μL cleared lysate.		#Save 20 μL 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.
	#*''I typically repeat this step to load the rest of my cleared lysate.''		#*''I typically 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.
	#*''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.
	#*''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.
	#*''Most of the protein should elute in this elution step.''		#*''Most of the protein should elute in this elution step.''