Knight:Centrifuge desalting/Sephadex columns: Difference between revisions
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==Overview== | ==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]] | |||
==Procedure== | |||
===Preparing the gel=== | |||
#Mix an appropriate weight of dry Sephadex powder with excess [[Knight:Protein DNA binding buffer|protein DNA binding buffer]]. | |||
#*''Protocol calls for usig 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.'' | |||
#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? | |||
#Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column. | |||
#Readjust the column to the vertical position. | |||
==References== | ==References== | ||
| Line 9: | Line 28: | ||
#Saul-AnalBiochem-1984 pmid=6204553 | #Saul-AnalBiochem-1984 pmid=6204553 | ||
#Helmerhorst-AnalBiochem-1980 pmid=6247935 | #Helmerhorst-AnalBiochem-1980 pmid=6247935 | ||
#GelFiltration ''Gel filtration: Principles and Methods'' by Amersham Pharmacia Biotech [http://www4.amershambiosciences.com/aptrix/upp00919.nsf/(FileDownload)?OpenAgent&docid=6EEE47990D9F933EC1256F90000DD697&file=18102218AI.pdf pdf] (contains a lot of practical, detailed instructions) | |||
</biblio> | </biblio> | ||
Revision as of 14:03, 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
Procedure
Preparing the gel
- Mix an appropriate weight of dry Sephadex powder with excess protein DNA binding buffer.
- Protocol calls for usig 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?
- Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.
- Readjust the column to the vertical position.
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)
