To ensure that the protein is completely dissolved and that only one protein molecule is present in each detergent micelle, excess of detergent is often employed to solubilize membrane proteins. However, it may bring trouble to protein researchers, as excess detergent may not be desired to exist in the downstream applications. On the one hand, an excess amount of detergent presents in the samples may interfere with protein activity or concentration measurement. On the other hand, excess detergent can unfortunately complicate spectra or disrupt further experimental work. In other cases, the detergent initially used for solubilization may not be appropriate for subsequent analytical procedure. Therefore, removal of excessive or unwanted detergent is an essential step in membrane protein research.
Detergents Removal Strategies
The optimal approach for removal of detergent from protein samples depends upon the type of detergent present in the sample. In other words, corresponding methods will be used to remove detergent according to different types of detergent with different properties in aqueous solution such as critical micelle concentration (CMC), charge or the aggregation number. Generally, detergents with a high (>1 mM) CMC are easily removed by methods such as dialysis or through ultrafiltration membranes. Detergents with low (<1 mM) CMC should be chromatographic methods. At present, there are several commonly used detergents removal methods, including dialysis, hydrophobic adsorption, gel chromatography, ion-exchange chromatography, and nickel columns and His tags .
- Dialysis: Dialysis is the most common method of detergent removal. The method is available if the detergent micelles are small enough to pass through the dialysis tube as a whole, or if the detergent has high CMC so that relatively more detergent exists in monomeric rather than micellar form. Typically, this process requires dialysing the protein detergent mixtures with detergent-free buffer (in about 200-fold excess) over a period of days. The technique is unsuitable for detergents with a low CMC, for example, nonionic detergents.
Fig. 1. Dialysis methods for detergent removal.
- Hydrophobic adsorption: The method takes advantage of the ability of amphiphilic detergent to bind to insoluble hydrophobic resins or "beads". Through the interaction of hydrophobic detergent tail with the hydrophobic surface of the bead, the resins will bind detergents then it can be removed by centrifugation or filtration. This method is effective for removal of most detergents and especially suitable for the removal of detergents with a low CMC.
Fig. 2. Hydrophobic methods for detergent removal.
- Gel chromatography: This technique works on the basis of size separation. Due to the size differences between protein–detergent and detergent micelles or detergent–lipid micelles, the excess detergents can be removed. In this process, what should be paid attention to is the factors that can affect micellar size such as pH, temperature, and ionic strength should be kept constant to obtain reproducible results.
- Ion-exchange chromatography: Ion-exchange chromatography works by exploiting the difference in charge between protein–detergent micelles and homogeneous detergent vesicles. The technology can bind the protein to a column, wash away excess detergent, and elute with a suitable low-detergent buffer.
- Nickel columns and His tags: The His-tagged protein in detergent micelles can bind to the Nickel column, and excess detergents can be removed or exchanged by washing the column with another detergent micellar solution prior to elution off the column.
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- Seddon A. M., et al. Membrane proteins, lipids and detergents: not just a soap opera[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2004, 1666(1-2): 105-117.
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