Knight:Centrifuge desalting/Sephadex columns

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

1. 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.
2. 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

1. • 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.
2. 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?
3. Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.
4. Readjust the column to the vertical position.

References

1. 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 |
2. 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 |

3. Gel filtration: Principles and Methods by Amersham Pharmacia Biotech pdf (contains a lot of practical, detailed instructions)