Knight:Centrifuge desalting/Sephadex columns

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

Materials

 * Sephadex G-25 superfine
 * Knight:Protein DNA binding buffer
 * Knight:Protein DNA binding buffer supplemented with BSA to a concentration of 1mg/mL
 * Polyethylene disk
 * Column for Sephadex
 * Collection tube

Preparing the gel

 * 1) Mix an appropriate weight of dry Sephadex powder with excess protein DNA binding buffer.
 * 2) *Protocol calls for using 25mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work. Helmerhorst-AnalBiochem-1980
 * 3) *Bed volume is 4-6mL per gram of Sephadex G25 superfine.
 * 4) *''Try 1g sephadex per 40 mL protein DNA binding buffer.
 * 5) Allow Sephadex to hydrate by doing one of the following
 * 6) Incubate overnight at 20&deg;C (minimum time is 3 hours).
 * 7) *Swelling time depends on type of Sephadex. Minimum incubation time assumes Sephadex G-25 superfine.
 * 8) Autoclave 20 mins.
 * 9) *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.
 * 3) *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.
 * 4) *How necessary is this? Not included in all protocols.
 * 5) Break off tab at the bottom of empty column.
 * 6) Place empty column in 2mL collection tube.
 * 7) Tilt the column and pipette the well-mixed gel suspension down the inside wall of the column.
 * 8) *The suspension seemed to settle a lot. So I removed excess buffer and added more suspension.
 * 9) Readjust the column to the vertical position.
 * 10) Place a polyethylene disk on the surface of the gel and compress gel further to avoid air gap formation between gel and disk.
 * 11) *Ensures even loading of sample.
 * 12) *I omitted this step and it seemed okay.
 * 13) 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 400&mu;L protein DNA binding buffer.
 * 4) *Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work. Helmerhorst-AnalBiochem-1980
 * 5) Centrifuge at 1400 g for 15 secs.
 * 6) Discard flow through.
 * 7) Wash with 400&mu;L protein DNA binding buffer.
 * 8) *Protocol calls for using 100mM potassium phosphate, pH 8. But I think that my protein DNA binding buffer should work. Helmerhorst-AnalBiochem-1980
 * 9) Centrifuge at 1400 g for 15 secs.
 * 10) Discard flow through.
 * 11) Wash with 400&mu;L protein DNA binding buffer supplemented with BSA to a concentration of 1mg/mL.
 * 12) Centrifuge at 1400 g for 2 mins.
 * 13) 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.
 * 4) *''Reduce spin time to 30 secs to eliminate protein dilution by 20%.
 * 5) *Recovered very small volume with 30 secs spin.
 * 6) Sample should be in tube.