Knight:Centrifuge desalting/Sephadex columns

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in progress! very rough. may contain errors.

Contents

Overview

A method for buffer exchange of protein samples in small volume (100-200 μL) containing 10ng-5mg of protein using a microcentrifuge.

Materials

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

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

References

  1. Helmerhorst E and Stokes GB. . pmid:6247935. PubMed HubMed [Helmerhorst-AnalBiochem-1980]
  2. Saul A and Don M. . pmid:6204553. PubMed HubMed [Saul-AnalBiochem-1984]
  3. Gel filtration: Principles and Methods by Amersham Pharmacia Biotech pdf (contains a lot of practical, detailed instructions)

    [GelFiltration]

All Medline abstracts: PubMed HubMed
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