Knight:Centrifuge desalting/Sephadex columns

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<font color=red>in progress! very rough.  may contain errors.</font>
 
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==Overview==
==Overview==
A method for buffer exchange of protein samples in small volume (100-200 &mu;L) containing 10ng-5mg of protein using a microcentrifuge.
A method for buffer exchange of protein samples in small volume (100-200 &mu;L) containing 10ng-5mg of protein using a microcentrifuge.
Line 6: Line 4:
==Materials==
==Materials==
*Sephadex G-25 superfine
*Sephadex G-25 superfine
-
*[[Knight:Protein DNA binding buffer]]
+
*[[Knight:Protein DNA binding|Knight:Protein DNA binding buffer]]
-
*[[Knight:Protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL
+
*[[Knight:Protein DNA binding|Knight:Protein DNA binding buffer]] supplemented with BSA to a concentration of 1mg/mL
*Polyethylene disk
*Polyethylene disk
 +
*Column for Sephadex
 +
*Collection tube
==Procedure==
==Procedure==
Line 15: Line 15:
#*''Protocol calls for using 25mM potassium phosphate, pH 8.  But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
#*''Protocol calls for using 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.''
#*''Bed volume is 4-6mL per gram of Sephadex G25 superfine.''
-
#Incubate overnight at 20&deg;C (minimum time is 3 hours).   
+
#*''Try 1g sephadex per 40 mL [[Knight:Protein DNA binding|protein DNA binding buffer]].
-
#*''Swelling time depends on type of Sephadex.  Minimum incubation time assumes Sephadex G-25 superfine.''
+
#Allow Sephadex to hydrate by doing one of the following
 +
##Incubate overnight at 20&deg;C (minimum time is 3 hours).   
 +
##*''Swelling time depends on type of Sephadex.  Minimum incubation time assumes Sephadex G-25 superfine.''
 +
##Autoclave 20 mins.
 +
##*''This has the advantage of sterilizing the Sephadex slurry thereby avoiding fungus growth during long term storage.''
===Packing a column===
===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.
+
 
 +
#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 (i.e. 3/4 gel and 1/4 buffer).  Fine particles can be removed by decantation if desired.
#Degas suspension under vacuum.
#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.''
#*''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?  Not included in all protocols.''
#*''How necessary is this?  Not included in all protocols.''
-
#Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.   
+
#Break off tab at the bottom of empty column.
 +
#Place empty column in 2mL collection tube.
 +
#Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.
 +
#*''The suspension seemed to settle a lot.  So I removed excess buffer and added more suspension.''  
#Readjust the column to the vertical position.   
#Readjust the column to the vertical position.   
#Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.
#Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.
#*''Ensures even loading of sample.''
#*''Ensures even loading of sample.''
 +
#*''I omitted this step and it seemed okay.''
#It is unclear what bed volume to aim for.
#It is unclear what bed volume to aim for.
===Column equilibration===
===Column equilibration===
-
#Remove stoppers from column.
+
#Pipette out excess buffer from column.
-
#Aspirate excess buffer from column.
+
#Centrifuge at 1400 ''g'' for 2 mins.
#Centrifuge at 1400 ''g'' for 2 mins.
-
#Wash with 1mL [[Knight:Protein DNA binding|protein DNA binding buffer]].
+
#Wash with 400&mu;L [[Knight:Protein DNA binding|protein DNA binding buffer]].
#*''Protocol calls for using 100mM potassium phosphate, pH 8.  But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
#*''Protocol calls for using 100mM potassium phosphate, pH 8.  But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
#Centrifuge at 1400 ''g'' for 15 secs.
#Centrifuge at 1400 ''g'' for 15 secs.
#Discard flow through.
#Discard flow through.
-
#Wash with 1mL [[Knight:Protein DNA binding|protein DNA binding buffer]].
+
#Wash with 400&mu;L [[Knight:Protein DNA binding|protein DNA binding buffer]].
#*''Protocol calls for using 100mM potassium phosphate, pH 8.  But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
#*''Protocol calls for using 100mM potassium phosphate, pH 8.  But I think that my protein DNA binding buffer should work.''<cite>Helmerhorst-AnalBiochem-1980</cite>
#Centrifuge at 1400 ''g'' for 15 secs.
#Centrifuge at 1400 ''g'' for 15 secs.
#Discard flow through.
#Discard flow through.
-
#Wash with 1mL [[Knight:Protein DNA binding|protein DNA binding buffer]] supplemented with extra BSA to a concentration of 1mg/mL.   
+
#Wash with 400&mu;L [[Knight:Protein DNA binding|protein DNA binding buffer]] supplemented with BSA to a concentration of 1mg/mL.   
#Centrifuge at 1400 ''g'' for 2 mins.
#Centrifuge at 1400 ''g'' for 2 mins.
#Discard flow through.
#Discard flow through.
Line 50: Line 58:
#Centrifuge at 1400 ''g'' for 2 mins.
#Centrifuge at 1400 ''g'' for 2 mins.
#*''Reduce spin time to 30 secs to eliminate protein dilution by 20%.
#*''Reduce spin time to 30 secs to eliminate protein dilution by 20%.
 +
#*''Recovered very small volume with 30 secs spin.''
#Sample should be in tube.
#Sample should be in tube.
-
====Notes====
+
==Notes==
-
*'''[[User:Rshetty|Reshma]] 17:32, 26 September 2006 (EDT)''': Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.
+
*Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.<cite>Helmerhorst-AnalBiochem-1980</cite> 
-
#Columns can be reused up to 6 times.
+
**''Haven't tried this myself.''
 +
*Columns can be reused up to 6 times by repeating column equilibration steps described above (except removal of excess buffer). <cite>Helmerhorst-AnalBiochem-1980</cite>
 +
**''Haven't tried this myself.''
==References==
==References==

Current revision

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 using 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.
    • Try 1g sephadex per 40 mL protein DNA binding buffer.
  2. Allow Sephadex to hydrate by doing one of the following
    1. 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.
    2. Autoclave 20 mins.
      • This has the advantage of sterilizing the Sephadex slurry thereby avoiding fungus growth during long term storage.

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 (i.e. 3/4 gel and 1/4 buffer). 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? Not included in all protocols.
  3. Break off tab at the bottom of empty column.
  4. Place empty column in 2mL collection tube.
  5. Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.
    • The suspension seemed to settle a lot. So I removed excess buffer and added more suspension.
  6. Readjust the column to the vertical position.
  7. Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.
    • Ensures even loading of sample.
    • I omitted this step and it seemed okay.
  8. It is unclear what bed volume to aim for.

Column equilibration

  1. Pipette out excess buffer from column.
  2. Centrifuge at 1400 g for 2 mins.
  3. Wash with 400μL protein DNA binding buffer.
    • Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.[1]
  4. Centrifuge at 1400 g for 15 secs.
  5. Discard flow through.
  6. Wash with 400μL protein DNA binding buffer.
    • Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work.[1]
  7. Centrifuge at 1400 g for 15 secs.
  8. Discard flow through.
  9. Wash with 400μL protein DNA binding buffer supplemented with BSA to a concentration of 1mg/mL.
  10. Centrifuge at 1400 g for 2 mins.
  11. Discard flow through.

Running the column

  1. Move column to new tube.
  2. Apply sample to column.
  3. Centrifuge at 1400 g for 2 mins.
    • Reduce spin time to 30 secs to eliminate protein dilution by 20%.
    • Recovered very small volume with 30 secs spin.
  4. Sample should be in tube.

Notes

  • Stopped columns equilibrated in 100mM potassium phosphate, pH8 containing 0.2% sodium azide can be stored at room temperature for several months.[1]
    • Haven't tried this myself.
  • Columns can be reused up to 6 times by repeating column equilibration steps described above (except removal of excess buffer). [1]
    • Haven't tried this myself.

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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