Knight:Centrifuge desalting/Sephadex columns
< Knight:Centrifuge desalting
in progress! very rough. may contain errors.
A method for buffer exchange of protein samples in small volume (100-200 μL) containing 10ng-5mg of protein using a microcentrifuge.
- Sephadex G-25 superfine
- Knight:Protein DNA binding buffer
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.
- 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.