Knight:Centrifuge desalting/Sephadex columns: Difference between revisions
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*Sephadex G-25 superfine | *Sephadex G-25 superfine | ||
*[[Knight:Protein DNA binding buffer]] | *[[Knight:Protein DNA binding buffer]] | ||
*[[Knight:Protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL | |||
*Polyethylene disk | |||
==Procedure== | ==Procedure== | ||
===Preparing the gel=== | ===Preparing the gel=== | ||
#Mix an appropriate weight of dry Sephadex powder with excess [[Knight:Protein DNA binding | #Mix an appropriate weight of dry Sephadex powder with excess [[Knight:Protein DNA binding|protein DNA binding buffer]]. | ||
#*''Protocol calls for | #*''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). | #Incubate overnight at 20°C (minimum time is 3 hours). | ||
Line 20: | Line 22: | ||
#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? | #*''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. | #Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column. | ||
#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. | |||
#*''Ensures even loading of sample.'' | |||
#It is unclear what bed volume to aim for. | |||
===Column equilibration=== | |||
#Remove stoppers from column. | |||
#Aspirate excess buffer from column. | |||
#Centrifuge at 1400 ''g'' for 2 mins. | |||
#Wash with 1mL [[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> | |||
#Centrifuge at 1400 ''g'' for 15 secs. | |||
#Discard flow through. | |||
#Wash with 1mL [[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> | |||
#Centrifuge at 1400 ''g'' for 15 secs. | |||
#Discard flow through. | |||
#Wash with 1mL [[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. | |||
#Discard flow through. | |||
===Running the column=== | |||
#Move column to new tube. | |||
#Apply sample to column. | |||
#Centrifuge at 1400 ''g'' for 2 mins. | |||
#*''Reduce spin time to 30 secs to eliminate protein dilution by 20%. | |||
#Sample should be in tube. | |||
====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. | |||
#Columns can be reused up to 6 times. | |||
==References== | ==References== |
Revision as of 14:32, 26 September 2006
in progress! very rough. may contain errors.
Overview
A method for buffer exchange of protein samples in small volume (100-200 μL) containing 10ng-5mg of protein using a microcentrifuge.
Materials
- Sephadex G-25 superfine
- Knight:Protein DNA binding buffer
- Knight:Protein DNA binding buffer supplemented with extra BSA to a concentration of 1mg/mL
- Polyethylene disk
Procedure
Preparing the gel
- Mix an appropriate weight of dry Sephadex powder with excess protein DNA binding buffer.
- Protocol calls for using 25mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.[1]
- Bed volume is 4-6mL per gram of Sephadex G25 superfine.
- 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.
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.
- 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.
- 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.
- 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.
- Ensures even loading of sample.
- It is unclear what bed volume to aim for.
Column equilibration
- Remove stoppers from column.
- Aspirate excess buffer from column.
- Centrifuge at 1400 g for 2 mins.
- Wash with 1mL protein DNA binding buffer.
- Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.[1]
- Centrifuge at 1400 g for 15 secs.
- Discard flow through.
- Wash with 1mL protein DNA binding buffer.
- Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.[1]
- Centrifuge at 1400 g for 15 secs.
- Discard flow through.
- Wash with 1mL protein DNA binding buffer supplemented with extra BSA to a concentration of 1mg/mL.
- Centrifuge at 1400 g for 2 mins.
- Discard flow through.
Running the column
- Move column to new tube.
- Apply sample to column.
- Centrifuge at 1400 g for 2 mins.
- Reduce spin time to 30 secs to eliminate protein dilution by 20%.
- Sample should be in tube.
Notes
- 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.
- Columns can be reused up to 6 times.
References
- Helmerhorst E and Stokes GB. Microcentrifuge desalting: a rapid, quantitative method for desalting small amounts of protein. Anal Biochem. 1980 May 1;104(1):130-5. DOI:10.1016/0003-2697(80)90287-0 |
- Saul A and Don M. A rapid method of concentrating proteins in small volumes with high recovery using Sephadex G-25. Anal Biochem. 1984 May 1;138(2):451-3. DOI:10.1016/0003-2697(84)90838-8 |
-
Gel filtration: Principles and Methods by Amersham Pharmacia Biotech pdf (contains a lot of practical, detailed instructions)