Knight:Centrifuge desalting/Sephadex columns
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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
- Knight:Protein DNA binding buffer supplemented with BSA to a concentration of 1mg/mL
- Polyethylene disk
- Column for Sephadex
- Collection tube
Procedure
Preparing the gel
- 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.
- Allow Sephadex to hydrate by doing one of the following
- 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.
- Autoclave 20 mins.
- This has the advantage of sterilizing the Sephadex slurry thereby avoiding fungus growth during long term storage.
- Incubate overnight at 20°C (minimum time is 3 hours).
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 (i.e. 3/4 gel and 1/4 buffer). Fine particles can be removed by decantation if desired.
- 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.
- 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.
- 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.
- It is unclear what bed volume to aim for.
Column equilibration
- Pipette out excess buffer from column.
- Centrifuge at 1400 g for 2 mins.
- 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]
- Centrifuge at 1400 g for 15 secs.
- Discard flow through.
- 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]
- Centrifuge at 1400 g for 15 secs.
- Discard flow through.
- Wash with 400μL protein DNA binding buffer supplemented with BSA to a concentration of 1mg/mL.
- Centrifuge at 1400 g for 2 mins.
- Discard flow through.
Running the column
- Move column to new tube.
- Apply sample to column.
- 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.
- 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
- 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 |
- 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 |
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Gel filtration: Principles and Methods by Amersham Pharmacia Biotech pdf (contains a lot of practical, detailed instructions)