Proteins that are bound strongly to the hydrophobic portion of cell membranes require detergents to facilitate dissociation. Because detergents can interfere with many downstream applications, detergent removal may be necessary after initially using them to extract or purify protein. Several different detergent removal methods are available: gel filtration, dialysis, Extracti-Gel® D Detergent Removing Gel (Product No. 20208, 20346), SDS-Out™ SDS Precipitation Reagent (Product No. 20308, 20310) and ion-exchange chromatography. Which method is appropriate depends on the effective molecular weight, concentration and other properties of the detergent. No one method is appropriate for all situations.
Overview of Removal Methods
Gel filtration (e.g., D-Salt™ Desalting Columns or Zeba™ Micro Desalt Spin Columns) removes detergents by size exclusion. Detergent monomers remain in the internal pores of the gel, and the protein is free to pass through the in the void volume. Pierce offers desalting columns with excellulose, dextran, polyacrylamide, and other matrices. Dialysis removes detergents by size exclusion but takes more time than gel filtration. Slide-A-Lyzer® Dialysis Cassettes (e.g., Product No. 66382), which can reduce dialysis time and provide excellent sample recovery, are available for several sample volumes and with 10 kD, 7 kD and 3.5 kD molecular weight cutoff membranes. Slide-A-Lyzer MINI Dialysis Units (e.g., Product No. 69576) are also available for dialyzing very small (10-100 μl) sample volumes. Extracti-Gel® D Detergent Removing Gel and the SDS-Out™ SDS Precipitation Reagent and Kit are quick, convenient methods for removing detergents that cannot be removed by either dialysis or gel filtration. Extracti-Gel® D Detergent Removing Gel is effective for binding and removing milligram quantities of many detergents from protein solutions. The SDS-Out™ Reagent is specific for removing SDS, a commonly used anionic detergent. Ion-exchange chromatography will remove nonionic and zwitterionic detergents. In this method, the protein is adsorbed on the resin and the detergent micelles pass through. Changing either the ionic strength or the pH can then elute the protein. Specific binding and elution procedures must be determined empirically for each protein being purified in this manner.
General Detergent Properties
Because the physical properties of detergents affect how easily they can be removed from a sample, these properties must be understood before choosing which removal method is appropriate. Micelles are associations of many detergent monomers that form spontaneously in solution. The critical micelle concentration (CMC) of a detergent is the minimum concentration at which micelles form; above the CMC, a detergent exists a in a large molecular weight association. The CMC is also an indicator of the strength at which detergent binds to protein; i.e., low values indicate strong binding and high values indicate weak binding. The CMC is also an indication of a detergent’s hydrophilicity. For most detergent to be removed by dialysis or gel filtration, the detergent concentration must be less than the CMC because only detergent monomers can be removed by these methods. A few detergents (e.g., CHAPS and Octyl-ß-Glucoside) have low molecular weight micelles (<10,000) and may be removed by dialysis or gel filtration even when the CMC has been exceeded. Table 1 indicates the detergent concentrations that can be removed by dialysis or desalting. When detergent removal by size exclusion is desired, choose a detergent with a high CMC and a low micelle molecular weight (e.g., Octyl-ß-Glucoside). Conversely, detergents with a low CMC and a high molecular weight (e.g., Triton X-100) are very difficult to remove from solution.