While detergents can be used to extract, solubilize, and manipulate (disrupt or form) membrane proteins from biological membranes for subsequent biochemical and physical characterization, and are useful in controlling protein crystallization and preventing nonspecific binding in affinity purification and immunoassay procedures, they can also be one of your greatest foes in the laboratory.
Aside from its role in protein solubilization, detergents also play an important function in cell lysis, protein crystallization, electrophoresis, and in the prevention of non-specific binding in affinity purification and immunoassay procedures.
Using Detergents for Protein Extraction: Some Important Things to Consider
Choose the right detergent for the job. To get the kind of results you want, you should be able to choose the most suitable detergent for your application. For example, you should choose the non-denaturing non-ionic detergents (Triton, Tween and Brij) if you merely want to break lipid-lipid and lipid-protein interactions without disrupting native interactions and structures of water-soluble proteins.
Ionic detergents (SDS, CTAB, etc.) should be used if your application calls for the complete disruption of cellular structure and denaturation of proteins while zwitterionic detergents (CHAPS, FOS-Choline 12, LDAO) are ideally used for chromatography, mass spectrometry, isoelectric focusing and 2D electrophoresis as well as for solubilization of organelles and inclusion bodies since they do not alter the native charge of the protein molecules.
Special note when using ionic detergents: These detergents are more sensitive to pH, ionic strength, and the nature of the counter ion than any other detergents. They are also known to interfere with downstream charge-based analytical methods.
Use proteomic grade detergents. Many commercial grade detergents contain elevated levels of sulfhydryl oxidizing agents, peroxides, salts and carbonyl compounds. The presence of these harmful reagents, particularly the peroxides and carbonyls (aldehydes), can increase over time as the product undergoes degradation. When this happens, they may react with protein amino acids causing modifications to the protein's primary structure and molecular mass. Worse, they may also inhibit protein-protein interactions.
To reduce the risk of artifactual modifications to the protein and preserve the integrity of your precious sample, it is recommended that you use proteomic grade detergents that contain low peroxide and aldehye contaminants. For best results, choose detergents that are sealed under inert gas to prevent oxidation upon exposure.
Determine the required degree of detergent purity and homogeneity. Certain applications require different degrees of purity and homogeneity. For example, a detergent that is both pure (free of contaminating alcohols, amides, or other by-products of synthesis) and homogeneous (composed of a single species) is needed when purifying and/or crystallizing proteins so commercial grade detergents should not be used for this purpose.
Consider efficiency of extracting the protein. Generally, you should choose a detergent based on the solubilization capability of your protein of interest.
Consider ease of removal. When choosing a detergent, it is also important to take the ease of removal or exchange into account since excess or unwanted detergent may interfere with certain applications and must be removed when reconstituting into liposomes. Keep in mind that detergents with high CMCs (e.g. octylglucoside, the MEGA-series of detergents, CHAPS, sodium cholate) are easily removed by dialysis while those with low CMCs are typically removed by adsorption to hydrophobic beads.
Some special precautions:
- Avoid using Triton® X-100 if the absorbance at 280 nm is an important parameter in your application.
- Avoid using charged detergents if the separation technique is based on charge differences (e.g. ion exchange chromatography or electrophoresis).
- Don't use detergents that form insoluble complexes (including all bile salts such as sodium cholate with its carboxylic acid group Lauroylsarcosinate) if divalent cations are essential for function.
- Stick to detergents with known physical parameters if you need to obtain precise physical data.
Image source: University of Michigan
