Protein Dialysis, Desalting, and Concentration Support—Troubleshooting

All molecules have different diffusion rates across membranes and may not have acted as other compounds of similar molecular weight. Increase dialysis time and/or number of buffer exchanges performed, or alternatively, use a device containing a higher molecular weight cut-off membrane. 

Water moves quickly and easily across the dialysis membrane. When dialyzing a high solute concentration against a dilute dialysis buffer, there will be a net movement of water (and possibly salts) into the dialysis unit through the membrane. Glycerol and some sugars are especially hygroscopic, and as rapidly as they diffuse across the membrane to reach equilibrium, they also significantly affect the osmosis of water across the membrane and so may cause a change in volume of the sample. Take care when dialyzing with large differences in glycerol or sugar concentration between sample and dialysis membrane. Prevent this movement of water and consequent change in sample volume by dialyzing in a “stepwise” fashion, minimizing the difference in water concentration between sample and dialysis buffer at each stage in the dialysis process (see more in Dialysis: An Overview). 

The smallest sizes of the Slide-A-Lyzer™ Mini Dialysis Units (10–100 μL) are inserted into a float. If the top of the sample is below the top of the buffer, then hydrostatic pressure of the buffer will force buffer into the unit, increasing the volume and diluting the sample. Making sure that the dialysis membrane is in contact with the buffer and the top of the sample is at or above the level of the buffer will prevent this 

The dialysis membranes are made of regenerated cellulose, which may cause some molecules to stick nonspecifically resulting in sample loss. The percent of total protein lost is partially dependent on the protein concentration. Protein loss caused by nonspecific binding to the membrane is negligible for concentrated samples (>0.5 mg/mL) but may be significant with dilute protein samples (<0.1 mg/mL). Adding a “carrier” protein such as BSA to dilute protein sample before dialysis will prevent this loss.

You can either reduce the volume of sample processed in each column (the product manual indicates the volume range recommended for each column size) or run the flow-through from one column over a fresh column.

Glycerol and sugars add viscosity to a sample, while detergents create micelles, making the removal of each difficult. Although dialysis is a better choice for removing these components, Zeba™ Columns can be used by modifying the desalting protocol. Modifications include diluting the samples, processing smaller samples, and adding the sample slowly to the column allowing it to enter the gel and equilibrate into the pores before centrifugation.

In order to obtain a high protein recovery (> 90%) when using the PES concentrators, make sure your protein of interest has at least a 2-fold higher molecular weight than the MWCO of the concentrator. For example, use a 5 K MWCO device for a >10 kDa protein.

There are different ways to concentrate your protein. These include:

• Centrifugation or filtration using concentrator columns or cassettes, respectively
• Dialysis
• Lyophilization
• Precipitation