Detergent Properties and Applications

By: Vicki Caligur, BioFiles 2008, 3.3, 14.

The key to detergent function is an amphipathic structure. All detergents are characterized as containing a hydrophilic “head” region and a hydrophobic “tail” region (see Figure 1).

Figure 1. Structure of the anionic detergent sodium dodecyl sulfate (SDS), showing the hydrophilic and hydrophobic regions.

These structural characteristics allow detergents to aggregate in aqueous media. At a sufficiently high concentration, the polar hydrophilic region of each molecule is oriented toward the polar solute (water) while the hydrophobic regions are grouped together to form thermodynamically stable micelles with hydrophobic cores. The hydrophobic core region of the detergent micelle associates with the hydrophobic surfaces of proteins and results in soluble protein-detergent complexes. Figure 2 is a simple illustration of a micelle to demonstrate the orientation concept. Actual micelle structures are more complex and dynamic, and can change due to detergent concentration and solution composition.1


Figure 2. Simple illustration of a sodium dodecyl sulfate micelle.

Biological detergents are commonly used to disrupt the bipolar lipid membrane of cells in order to release and solubilize membrane-bound proteins. Some detergents can be used to solubilize recombinant proteins, while others are recommended for the stabilization, crystallization, or denaturation of proteins. Detergents can align at aqueous/non-aqueous interfaces, resulting in reduced surface tension, increased miscibility, and stabilization of emulsions. Additional detergent applications include:

  • Extraction of DNA and RNA
  • Solubilization of specimens for diagnostic applications
  • Cell lysis
  • Liposome preparation
  • Prevention of reagent and analyte precipitation from solution
  • Prevention of non-specific binding in immunoassays

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Detergent Physical Characteristics

The concentration at which micelles begin to form is the critical micelle concentration (CMC). The CMC is the maximum monomer concentration and constitutes a measure of the free energy of micelle formation. The lower the CMC, the more stable the micelle and the more slowly molecules are incorporated into or removed from the micelle. The structure of the hydrophobic region of the detergent can affect the micelle structure. An increase in the length of the hydrophobic hydrocarbon chain of ionic detergents results in an increased micelle size and a lower CMC, as fewer molecules are needed to construct a micelle.

The average number of monomers in a micelle is the aggregation number. The CMC and aggregation number values are highly dependent on factors such as temperature, pH, ionic strength, and detergent homogeneity and purity. Slight discrepancies in reported values for CMC and aggregation number may be the result of variations in the analytical methods used to determine the values. Aggregation number values are also shifted by concentration, since the number of detergent molecules per micelle may increase if the concentration is above the CMC.

Ease of removal or exchange is an important factor in the selection of a detergent. Some of the more common detergent removal methods include:

  • Dialysis
  • Gel filtration chromatography
  • Hydrophobic adsorption chromatography
  • Protein precipitation


The CMC value associated with the detergent is a useful guide to hydrophobic binding strength. Detergents with higher CMC values have weaker binding and are subsequently easier to remove by dialysis or displacement methods. Detergents with low CMC values require less detergent in order to form micelles and solubilize proteins or lipids.

Another useful parameter when evaluating detergents for downstream removal is the micelle molecular weight, which indicates relative micelle size. Smaller micelles are more easily removed and are usually desirable when protein-detergent complexes are to be separated based on the molecular size of the protein. The micelle molecular weight may be calculated by multiplying the aggregation number by the monomer molecular weight.

The cloud point is the temperature at which the detergent solution near or above its CMC separates into two phases. The micelles aggregate, typically forming a cloudy phase with high detergent concentration, while the balance of the solution becomes detergent-depleted. The resulting two-phase solution can be separated, with the extracted protein being located in the detergent-rich phase. Detergents with low cloud point temperatures, such as TRITON® X-114 (cloud point ~23 °C) are recommended for use with proteins since high cloud point temperatures may denature solubilized proteins. The cloud point can be affected by changes in detergent concentration, temperature, and the addition of salt or polymers such as dextran and polyethylene glycol. Note that the detergent-rich phase is also contingent on the specific detergent(s) and salt concentration; under some conditions the phase may be clear rather than cloudy and be located as either the upper or lower phase of the solution. In non-ionic detergents, this behavior has been applied in the phase separation and purification of membrane proteins.2

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Detergent Types and Selection

When selecting a detergent, the first consideration is usually the form of the hydrophilic group:

  • Anionic
  • Cationic
  • Non-ionic
  • Zwitterionic (ampholytic)

Anionic and cationic detergents are considered biologically “harsh” detergents because they typically modify protein structure to a greater extent than neutrally charged detergents. The degree of denaturation varies with the individual protein and the particular detergent and concentration. Ionic detergents are more sensitive to pH, ionic strength, and the nature of the counter ion, and can interfere with downstream charge-based analytical methods.

Non-ionic detergents are considered to be “mild” detergents because they are less likely than ionic detergents to denature proteins. By not separating protein-protein bonds, non-ionic detergents allow the protein to retain its native structure and functionality, although detergents with shorter hydrophobic chain lengths are more likely to cause protein deactivation. Many nonionic detergents can be classified into three structure types:

  • Poly(oxyethylene) ethers and related polymers
  • Bile salts
  • Glycosidic detergents

Poly(oxyethylene) ethers and related detergents have a neutral, polar head and hydrophobic tails that are oxyethylene polymers (e.g. Brij® and TWEEN®) or ethyleneglycoether polymers (e.g. TRITON®). The tert-octylphenol poly(ethyleneglycoether) series of detergents, which includes TRITON X-100 and IGEPAL® CA-630, have an aromatic head that interferes with downstream UV analysis techniques.

Bile salts have a steroid core structure with a polar and apolar orientation, rather than the more obvious nonpolar tail structure of other detergents. Bile salts may be less denaturing than linear chain detergents with the same polar head group.

Glycosidic detergents have a carbohydrate, typically glucose or maltose, as the polar head and an alkyl chain length of 7-14 carbons as the polar tail.

Zwitterionic detergents have characteristics of both ionic and non-ionic detergent types. Zwitterionic detergents are less denaturing than ionic detergents and have a net neutral charge, similar to non-ionic detergents. They are more efficient than non-ionic detergents at disrupting protein-protein bonds and reducing aggregation. These properties have been used for chromatography, mass spectrometry, and electrophoresis methods, and solubilization of organelles and inclusion bodies.

Non-detergent sulfobetaines (NDSB), although not detergents, possess hydrophilic groups similar to those of zwitterionic detergents but with shorter hydrophobic chains. Sulfobetaines do not form micelles. They have been reported to improve the yield of membrane proteins when used with detergents and prevent aggregation of denatured proteins.

Detergents Properties and Applications (199 Kb PDF)

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The following references are recommended for further review of the properties and applications of detergents.

A Helenius, D R McCaslin, E Fries, C Tanford
Methods in Enzymology 1979-01-01
J M Neugebauer
Methods in Enzymology 1990-01-01
L M Hjelmeland
Methods in Enzymology 1990-01-01
F A Marston, D L Hartley
Methods in Enzymology 1990-01-01
L M Hjelmeland
Methods in Enzymology 1990-01-01
Annela M Seddon, Paul Curnow, Paula J Booth
Biochimica et Biophysica Acta 2004-11-03
Studying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successful solubilization or reconstitution of membrane proteins, which...Read More
Gilbert G Privé
Methods 2007-04-01
The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but a...Read More

Cited References

R M Garavito, S Ferguson-Miller
Journal of Biological Chemistry 2001-08-31
Detergents are invaluable tools for studying membrane proteins. However, these deceptively simple, amphipathic molecules exhibit complex behavior when they self-associate and interact with other molecules. The phase behavior and assembled structures of detergents are markedly influenced not only by their unique chemical and phys...Read More
Thomas Arnold, Dirk Linke
BioTechniques 2007-10-01
Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinatio...Read More
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