Glycoproteomics: Buffer Exchange Protocols (P0711)

1. Select a sample preparation spin column (high protein recovery) adequate for handling up to 15 µg of protein and 10 µl volume. Following manufacturer’s recommendations, equilibrate the column in buffer of choice. Apply sample, incubate for a few minutes. Spin to elute protein sample, discard column.

2. Select a small dialysis device suitable for volumes from 10 to 100 µl, follow manufacturer instructions and recommendations. Dialysis can be performed several times against a small volume of buffer, or one time against a large volume of buffer. The extent of dialysis depends on time, sample volume, temperature, and agitation. The nature of the molecules to be removed (size, charge) as well as the nature of the dialysis buffer (concentration, ionic strength, pH) will also affect this process.

3. Drop dialysis is an inexpensive method for buffer exchange (although it requires careful manipulation). Pour 50 ml of dialysis buffer into a Petri dish, float a nitrocellulose membrane filter (0.025 µM) gently on the surface of the buffer. Pipette the sample (10–100 µl) on the center of the filter very gently (do not touch the filter with the pipette tip as this can cause the filter to submerge into the buffer). Cover the Petri dish and let sample sit on filter for 1 hour at room temperature (do not leave sample for more than 1 hour). Using a pipette, very gently pull sample off of the filter and place it in a clean micro-centrifuge tube. Following drop dialysis, a small increase in sample volume may occur.

4. Select a centrifugal filter unit suitable for small sample volumes, follow manufacturer instructions. Membranes with low-protein binding are best to minimize protein loss. Do not over concentrate your sample, as your target protein might precipitate resulting in poor recovery.