Knight:Centrifuge desalting/Sephadex columns - Revision history

Reshma P. Shetty at 14:18, 16 October 2006

2006-10-16T14:18:13Z

←Older revision		Revision as of 14:18, 16 October 2006
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-	~~<font color=red>in progress! very rough. may contain errors.</font>~~
-
	==Overview==		==Overview==
	A method for buffer exchange of protein samples in small volume (100-200 μL) containing 10ng-5mg of protein using a microcentrifuge.		A method for buffer exchange of protein samples in small volume (100-200 μL) containing 10ng-5mg of protein using a microcentrifuge.

Reshma P. Shetty: Knight:Centrifuge desalting moved to Knight:Centrifuge desalting/Sephadex columns: multiple protocols

2006-10-16T14:14:28Z

Knight:Centrifuge desalting moved to Knight:Centrifuge desalting/Sephadex columns: multiple protocols

←Older revision

Revision as of 14:14, 16 October 2006

Reshma P. Shetty at 20:41, 12 October 2006

2006-10-12T20:41:54Z

←Older revision		Revision as of 20:41, 12 October 2006
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	*Sephadex G-25 superfine		*Sephadex G-25 superfine
	*[[Knight:Protein DNA binding\|Knight:Protein DNA binding buffer]]		*[[Knight:Protein DNA binding\|Knight:Protein DNA binding buffer]]
-	*[[Knight:Protein DNA binding\|Knight:Protein DNA binding buffer]] supplemented with ~~extra~~ BSA to a concentration of 1mg/mL	+	*[[Knight:Protein DNA binding\|Knight:Protein DNA binding buffer]] supplemented with BSA to a concentration of 1mg/mL
	*Polyethylene disk		*Polyethylene disk
	*Column for Sephadex		*Column for Sephadex
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	#Centrifuge at 1400 ''g'' for 15 secs.		#Centrifuge at 1400 ''g'' for 15 secs.
	#Discard flow through.		#Discard flow through.
-	#Wash with 400μL [[Knight:Protein DNA binding\|protein DNA binding buffer]] supplemented with ~~extra~~ BSA to a concentration of 1mg/mL.	+	#Wash with 400μL [[Knight:Protein DNA binding\|protein DNA binding buffer]] supplemented with BSA to a concentration of 1mg/mL.
	#Centrifuge at 1400 ''g'' for 2 mins.		#Centrifuge at 1400 ''g'' for 2 mins.
	#Discard flow through.		#Discard flow through.
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	==Notes==		==Notes==
-	*Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.<cite>Helmerhorst-AnalBiochem-1980</cite>'' ''Haven't tried this myself.''	+	*Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.<cite>Helmerhorst-AnalBiochem-1980</cite>
-	*Columns can be reused up to 6 times.<cite>Helmerhorst-AnalBiochem-1980</cite>'' ''Haven't tried this myself.''	+	**''Haven't tried this myself.''
		+	*Columns can be reused up to 6 times by repeating column equilibration steps described above (except removal of excess buffer). <cite>Helmerhorst-AnalBiochem-1980</cite>
		+	**''Haven't tried this myself.''

	==References==		==References==

Reshma P. Shetty at 14:39, 5 October 2006

2006-10-05T14:39:57Z

←Older revision		Revision as of 14:39, 5 October 2006
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	==Notes==		==Notes==
-	*Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.	+	*Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.<cite>Helmerhorst-AnalBiochem-1980</cite>'' ''Haven't tried this myself.''
-	*Columns can be reused up to 6 times.	+	*Columns can be reused up to 6 times.<cite>Helmerhorst-AnalBiochem-1980</cite>'' ''Haven't tried this myself.''

	==References==		==References==

Reshma P. Shetty: /* Running the column */

2006-10-02T16:12:05Z

Running the column

←Older revision		Revision as of 16:12, 2 October 2006
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	#Centrifuge at 1400 ''g'' for 2 mins.		#Centrifuge at 1400 ''g'' for 2 mins.
	#*''Reduce spin time to 30 secs to eliminate protein dilution by 20%.		#*''Reduce spin time to 30 secs to eliminate protein dilution by 20%.
		+	#*''Recovered very small volume with 30 secs spin.''
	#Sample should be in tube.		#Sample should be in tube.

Reshma P. Shetty: /* Packing a column */

2006-10-02T16:10:52Z

Packing a column

←Older revision		Revision as of 16:10, 2 October 2006
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	#Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.		#Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.
	#*''Ensures even loading of sample.''		#*''Ensures even loading of sample.''
		+	#*''I omitted this step and it seemed okay.''
	#It is unclear what bed volume to aim for.		#It is unclear what bed volume to aim for.

Reshma P. Shetty at 16:17, 28 September 2006

2006-09-28T16:17:10Z

←Older revision		Revision as of 16:17, 28 September 2006
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	===Packing a column===		===Packing a column===
-	#*Adjust the suspension of the gel so that it is a fairly thick slurry. It should not be so thick as to retain air bubbles. 75% of settled gel is suitable (i.e. 3/4 gel and 1/4 buffer). Fine particles can be removed by decantation if desired.	+
		+	#Adjust the suspension of the gel so that it is a fairly thick slurry. It should not be so thick as to retain air bubbles. 75% of settled gel is suitable (i.e. 3/4 gel and 1/4 buffer). Fine particles can be removed by decantation if desired.
	#Degas suspension under vacuum.		#Degas suspension under vacuum.
	#*''Not necessary if the Sephadex was swollen using a boiling water bath. The gel suspension should be allowed to cool to temperature of column operation however.''		#*''Not necessary if the Sephadex was swollen using a boiling water bath. The gel suspension should be allowed to cool to temperature of column operation however.''
	#*''How necessary is this? Not included in all protocols.''		#*''How necessary is this? Not included in all protocols.''
-	#Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.	+	#Break off tab at the bottom of empty column.
		+	#Place empty column in 2mL collection tube.
		+	#Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.
		+	#*''The suspension seemed to settle a lot. So I removed excess buffer and added more suspension.''
	#Readjust the column to the vertical position.		#Readjust the column to the vertical position.
	#Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.		#Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.
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	===Column equilibration===		===Column equilibration===
-	#~~Remove stoppers from column.~~	+	#Pipette out excess buffer from column.
-	~~#Aspirate~~ excess buffer from column.	+
	#Centrifuge at 1400 ''g'' for 2 mins.		#Centrifuge at 1400 ''g'' for 2 mins.
-	#Wash with ~~1mL~~ [[Knight:Protein DNA binding\|protein DNA binding buffer]].	+	#Wash with 400μL [[Knight:Protein DNA binding\|protein DNA binding buffer]].
	#*''Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>		#*''Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
	#Centrifuge at 1400 ''g'' for 15 secs.		#Centrifuge at 1400 ''g'' for 15 secs.
	#Discard flow through.		#Discard flow through.
-	#Wash with ~~1mL~~ [[Knight:Protein DNA binding\|protein DNA binding buffer]].	+	#Wash with 400μL [[Knight:Protein DNA binding\|protein DNA binding buffer]].
	#*''Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>		#*''Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
	#Centrifuge at 1400 ''g'' for 15 secs.		#Centrifuge at 1400 ''g'' for 15 secs.
	#Discard flow through.		#Discard flow through.
-	#Wash with ~~1mL~~ [[Knight:Protein DNA binding\|protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL.	+	#Wash with 400μL [[Knight:Protein DNA binding\|protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL.
	#Centrifuge at 1400 ''g'' for 2 mins.		#Centrifuge at 1400 ''g'' for 2 mins.
	#Discard flow through.		#Discard flow through.

Reshma P. Shetty: /* Packing a column */

2006-09-27T02:04:23Z

Packing a column

←Older revision		Revision as of 02:04, 27 September 2006
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	===Packing a column===		===Packing a column===
-	#*Adjust the suspension of the gel so that it is a fairly thick slurry. It should not be so thick as to retain air bubbles. 75% of settled gel is suitable. Fine particles can be removed by decantation if desired.	+	#*Adjust the suspension of the gel so that it is a fairly thick slurry. It should not be so thick as to retain air bubbles. 75% of settled gel is suitable (i.e. 3/4 gel and 1/4 buffer). Fine particles can be removed by decantation if desired.
	#Degas suspension under vacuum.		#Degas suspension under vacuum.
	#*''Not necessary if the Sephadex was swollen using a boiling water bath. The gel suspension should be allowed to cool to temperature of column operation however.''		#*''Not necessary if the Sephadex was swollen using a boiling water bath. The gel suspension should be allowed to cool to temperature of column operation however.''

Reshma P. Shetty: /* Preparing the gel */

2006-09-27T02:03:15Z

Preparing the gel

←Older revision		Revision as of 02:03, 27 September 2006
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	#*''Protocol calls for using 25mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>		#*''Protocol calls for using 25mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
	#*''Bed volume is 4-6mL per gram of Sephadex G25 superfine.''		#*''Bed volume is 4-6mL per gram of Sephadex G25 superfine.''
-	#Incubate overnight at 20°C (minimum time is 3 hours).	+	#*''Try 1g sephadex per 40 mL [[Knight:Protein DNA binding\|protein DNA binding buffer]].
-	#*''Swelling time depends on type of Sephadex. Minimum incubation time assumes Sephadex G-25 superfine.''	+	#Allow Sephadex to hydrate by doing one of the following
		+	##Incubate overnight at 20°C (minimum time is 3 hours).
		+	##*''Swelling time depends on type of Sephadex. Minimum incubation time assumes Sephadex G-25 superfine.''
		+	##Autoclave 20 mins.
		+	##*''This has the advantage of sterilizing the Sephadex slurry thereby avoiding fungus growth during long term storage.''

	===Packing a column===		===Packing a column===

Reshma P. Shetty at 21:57, 26 September 2006

2006-09-26T21:57:22Z

←Older revision		Revision as of 21:57, 26 September 2006
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	==Materials==		==Materials==
	*Sephadex G-25 superfine		*Sephadex G-25 superfine
-	*[[Knight:Protein DNA binding buffer]]	+	*[[Knight:Protein DNA binding\|Knight:Protein DNA binding buffer]]
-	*[[Knight:Protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL	+	*[[Knight:Protein DNA binding\|Knight:Protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL
	*Polyethylene disk		*Polyethylene disk
		+	*Column for Sephadex
		+	*Collection tube

	==Procedure==		==Procedure==
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	#Sample should be in tube.		#Sample should be in tube.

-	====Notes====	+	==Notes==
-	*~~'''[[User:Rshetty\|Reshma]] 17:32, 26 September 2006 (EDT)''':~~ Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.	+	*Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.
-	#Columns can be reused up to 6 times.	+	*Columns can be reused up to 6 times.

	==References==		==References==