The fastest way of buffer exchange is that offered by Bio-Spin 6 microcolumns (takes 25-30 mins), and it can be carried out right before spraying the sample. It is, therefore, advisable to try it first using the protocol below. Please note that we do not provide the Bio-Spin columns, you have to purchase them from the vendor.
Protocol for buffer exchange using Bio-Spin 6 microcolumns
Materials: ammonium acetate solution 50-200 mM, Bio-Spin 6 microcolumns with included waste vials, clean 2 ml Eppendorf vials, 100 and 1000 ml pipettes with tips, centrifuge for Eppendorf-type vials. Initial sample amount 25-80 ml required per single column. As the columns are centrifuged, a common practice is to make buffer exchange on two columns simultaneously counter-balancing them on the centrifuge. Protein concentration can be higher than the final 1-25 mM (please refer to Bio-Spin 6 instruction leaflet), and consequently diluted in ammonium acetate after the buffer exchange.
- Invert the column sharply several times to resuspend the settled gel and remove any bubbles.
- Snap off the tip and place column in a 2.0 ml microcentrifuge waste tube (included). Remove the cap. Allow the excess packing buffer to drain by gravity to the top of gel bed. (If the column does not begin to flow, push cap back into the column and remove). Discard the drained buffer, then place the column back into 2 ml waste tube.
- Centrifuge for 2 min in a swinging bucket centrifuge at 1,000 x g (to remove the packing buffer). Make sure you balance the column in the centrifuge, better by the same microcolumn/waste tube. Discard the buffer inn the waste tube.
- Put 500 ml of ammonium acetate buffer in the column, centrifuge 1 min at 1000xg, discard the buffer in the waste tube.
- Repeat step 4 three-five times discarding the buffer each time. The last time you can use 450 ml of ammonium acetate instead of 500 ml to avoid dilution of the sample in the step 6.
- Place the column in a clean 2.0 ml Eppendorf tube. Carefully apply the sample (25–80 µl) directly to the centre of the column. Application of more or less than the recommended sample volume may decrease column performance.
- After loading sample, centrifuge the column for 4 min at 1,000 x g. The sample in ammonium acetate will be at the bottom of the Eppendorf tube now.