Knight:Centrifuge desalting/Sephadex columns

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==Overview==
==Overview==
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A method for buffer exchange of protein samples in small volume (100-200 μL) containing 10ng-5mg of protein using a microcentrifuge.
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A method for buffer exchange of protein samples in small volumes using a microcentrifuge.
+
==Materials==
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*Sephadex G-25 superfine
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*[[Knight:Protein DNA binding buffer]]
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==Procedure==
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===Preparing the gel===
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#Mix an appropriate weight of dry Sephadex powder with excess [[Knight:Protein DNA binding buffer|protein DNA binding buffer]].
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#*''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>
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#*''Bed volume is 4-6mL per gram of Sephadex G25 superfine.''
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#Incubate overnight at 20&deg;C (minimum time is 3 hours). 
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#*''Swelling time depends on type of Sephadex.  Minimum incubation time assumes Sephadex G-25 superfine.''
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===Packing a column===
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#*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.
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#Degas suspension under vacuum.
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#*''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.''
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#*How necessary is this?
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#Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column. 
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#Readjust the column to the vertical position.
==References==
==References==
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#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 17:03, 26 September 2006

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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