A membrane protein is considered solubilized if it is present in the supernatant after one hour centrifugation of a lysate or a homogenate at 100,000 x g. In most cases, it is also important that the biological activity of the protein be preserved in the supernatant after solubilization by a detergent. Hence, the appropriate detergent should yield the maximum amount of biologically active protein in the supernatant. Given the large number of detergents available today, choosing an appropriate detergent can be a difficult process. Some of the points outlined below can be helpful in selecting a suitable detergent.
1. The first step is a survey of the literature. A detergent that has been used previously for the isolation and characterization of a protein with similar biochemical or enzymological properties should be tried first.
2. Solubility of the detergent at working temperature can be another consideration. For example, ZWITTERGENT® 3-14 is insoluble in water at +4°C while TRITON® X-114 undergoes a phase separation at room temperature.
3. The method of detergent removal can be an important consideration. If dialysis is to be employed, a detergent with a high CMC is clearly preferred. Alternatively, if ion exchange chromatography is utilized, a non-ionic detergent or a ZWITTERGENT® is the detergent of choice.
4. Preservation of biological or enzymological activity may require experimenting with several detergents. Not only the type but also the quantity of the detergent used will affect the activity of the protein. For some proteins biological activity is preserved over a very narrow range of concentration of detergent. Below this range the protein is not solubilized and above a particular concentration, the protein is inactivated.
5. Since TRITON® X-100 contains aromatic rings that absorb at 260-280 nm, this detergent should be avoided if the protocols require UV monitoring of protein concentration. Similarly, ionic detergents should be avoided if the proteins are to be separated by isoelectric focusing. For gel filtration of proteins, detergents with smaller aggregation numbers should be considered.
6. Detergents of utmost purity should be used since some detergents such as TRITON® X-100 are generally known to contain peroxides as contaminants. Calbiochem®’s PROTEIN GRADE® or ULTROL® GRADE detergents that have been purified to minimize these oxidizing contaminants are recommended.
7. Calbiochem® biochemicals also offers a variety of Molecular Biology Grade detergents for any research where contaminants such as DNase, RNase, and proteases are problematic.
8. A non-toxic detergent should be preferred over a toxic one. For example, digitonin, a cardiac glycoside, should be handled with special care.
9. For as yet unknown reasons, specific detergents often work better for particular isolation procedures. For example, n-Dodecyl-b-D-maltoside (Cat. No. 324355) has been found to be the detergent of choice for the isolation of cytochrome c oxidase. Hence, some “trial and error” may be required for determining optimal conditions for isolation of a membrane protein in its biologically active form.
10. In some cases, it has been observed that the inclusion of non-detergent sulfobetaines (NDSBs) with detergents in the isolation buffer dramatically improves yields of solubilized membrane proteins.
