Detergents are a class of molecules whose unique properties enable manipulation (disruption or formation) of hydrophobic-hydrophilic interactions among molecules in biological samples. In protein research, detergents are used to lyse cells (release soluble proteins), solubilize membrane proteins and lipids, control protein crystallization, prevent non-specific binding in affinity purification and immunoassay procedures, and are used as additives in electrophoresis.
Detergents are amphipathic molecules, meaning they contain both a non-polar "tail" having an aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for the broad classification of detergents; they may be ionic (charged, either anionic or cationic), non-ionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero).
Structure of surfactants. Generalized structure of a single detergent molecule (top) and the complete structure of CHAPS (bottom), an example of a zwitterionic detergent.
Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their non-polar tail groups. Nevertheless, detergents are themselves water-soluble. Consequently, detergent molecules allow the dispersion (miscibility) of water-insoluble, hydrophobic compounds into aqueous media, including the extraction and solubilization of membrane proteins.
Detergents at low concentration in an aqueous solution form a monolayer at the air-liquid interface. At higher concentrations, detergent monomers aggregate into structures called micelles. A micelle is a thermodynamically stable colloidal aggregate of detergent monomers wherein the non-polar ends are sequestered inward, avoiding exposure to water, and the polar ends are oriented outward in contact with the water.
Both the number of detergent monomers per micelle (aggregation number) and the range of detergent concentration above which micelles form (called the critical micelle concentration, CMC) are properties specific to each particular detergent (see table). The critical micelle temperature (CMT) is the lowest temperature at which micelles can form. The CMT corresponds to what is known as the cloud point since detergent micelles form crystalline suspensions at temperatures below the CMT and are clear again at temperatures above the CMT.
Detergent properties are affected by experimental conditions such as concentration, temperature, buffer pH and ionic strength, and the presence of various additives. For example, the CMC of certain non-ionic detergents decreases with increasing temperature, while the CMC of ionic detergents decreases with the addition of counter ion as a result of reduced electrostatic repulsion among the charged head groups. In other cases, additives such as urea effectively disrupt water structure and cause a decrease in detergent CMC. Generally, dramatic increases in aggregation number occur with increasing ionic strength.
Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking protein-protein interactions. Non-denaturing detergents can be divided into non-ionic detergents such as Triton X-100, bile salts such as cholate, and zwitterionic detergents such as CHAPS.
|Sodium dodecyl sulphate (SDS)||Anionic||Strong lysis agent. Works with majority of cells types. Not suitable for sensitive protein extraction due to denaturing properties.|
|Non-ionic||Non-denaturing, mild lysis agent. Works well when performing protein analysis.|
|NP-40||Non-ionic||Non-denaturing, mild lysis agent. Good for isolating cytoplasmic proteins but not nuclear proteins.|
|Non-ionic||Mild lysis agent. Good for cell lysis and protein isolation.|
|Zwitterionic||Mild lysis agent. Good for protein isolation.|
|Triton X-100||Non-ionic||140||647 (90K)||0.24 (0.0155)||64||No|
|Triton X-114||Non-ionic||–||537 ( – )||0.21 (0.0113)||23||No|
|NP-40||Non-ionic||149||617 (90K)||0.29 (0.0179)||80||No|
|Brij-35||Non-ionic||40||1225 (49K)||0.09 (0.0110)||>100||No|
|Brij-58||Non-ionic||70||1120 (82K)||0.08 (0.0086)||>100||No|
|Tween-20||Non-ionic||–||1228 ( – )||0.06 (0.0074)||95||No|
|Tween-80||Non-ionic||60||1310 (76K)||0.01 (0.0016)||–||No|
|Octyl-beta-Glucoside||Non-ionic||27||292 (8K)||23–24 (~0.70)||>100||Yes|
|Octylthio Glucoside||Non-ionic||–||308 ( – )||9 (0.2772)||>100||Yes|
|SDS (sodium dodecyl sulfate)||Anionic||62||288 (18K)||6–8 (0.17–0.23)||>100||No|
|CHAPS||Zwitterionic||10||615 (6K)||8–10 (0.5–0.6)||>100||Yes|
|CHAPSO||Zwitterionic||11||631 (7K)||8–10 (~0.505)||90||Yes|
‡Agg.# = Aggregation number, which is the number of molecules per micelle.
A major factor determining the behavior and interaction of molecules in biological samples is their hydrophilicity or hydrophobicity. Most proteins and other molecules with charged or polar functional groups are soluble (or miscible) in water because they participate in the highly ordered, hydrogen-bonded intermolecular structure of water. Some other proteins (or at least parts of proteins), as well as fats and lipids, lack polar or charged functional groups; consequently, they are excluded from the ordered interaction of water with other polar molecules and tend to associate together in structures having minimal surface area contact with the polar environment. This association of non-polar molecules in aqueous solutions is commonly called hydrophobic attraction, although it is more accurately understood as exclusion from the hydrophilic environment.
The formation and stability of biological membranes results in large measure from the hydrophobic attraction of phospholipids, which form bilayer sheets having hydrophobic lipid "tails" oriented within the sheet thickness and polar "head" groups oriented to the outer and inner aqueous environments. Membrane proteins completely span the membrane thickness or are embedded at one side of the membrane in accord with their structure of hydrophobic and hydrophilic amino acid side chains and other functional groups.
Generally, moderate concentrations of mild (i.e., non-ionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using certain buffer conditions, various detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins.
Detergent-based cell lysis. Both denaturing and non-denaturing cell lysis reagents may be used for protein extraction procedures.
Denaturing detergents such as SDS bind to both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins at concentrations below the CMC (i.e., as monomers). The reaction is equilibrium driven until saturated. Therefore, the free concentration of monomers determines the detergent concentration. SDS binding is cooperative (i.e., the binding of one molecule of SDS increases the probability that another molecule of SDS will bind to that protein) and alters most proteins into rigid rods whose length is proportional to molecular weight.
Non-denaturing detergents such as Triton X-100 have rigid and bulky non-polar heads that do not penetrate into water-soluble proteins; consequently, they generally do not disrupt native interactions and structures of water-soluble proteins and do not have cooperative binding properties. The main effect of non-denaturing detergents is to associate with hydrophobic parts of membrane proteins, thereby conferring miscibility to them.
At concentrations below the CMC, detergent monomers bind to water-soluble proteins. Above the CMC, binding of detergent to proteins competes with the self-association of detergent molecules into micelles. Consequently, there is effectively no increase in protein-bound detergent monomers with increasing detergent concentration beyond the CMC.
Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization. The initial stage is lysis or rupture of the membrane. At detergent:membrane lipid molar ratios of 0.1:1 through 1:1, the lipid bilayer usually remains intact but selective extraction of some membrane proteins occurs. Increasing the ratio to 2:1, solubilization of the membrane occurs, resulting in mixed micelles. These include phospholipid-detergent micelles, detergent-protein micelles, and lipid-detergent-protein micelles. At a ratio of 10:1, all native membrane lipid:protein interactions are effectively exchanged for detergent:protein interactions.
The amount of detergent needed for optimal protein extraction depends on the CMC, aggregation number, temperature, and nature of the membrane and the detergent. The solubilization buffer should contain sufficient detergent to provide greater than one micelle per membrane protein molecule to help ensure that individual protein molecules are isolated in separate micelles.
Detergents used for cell lysis. Major characteristics of denaturing and non-denaturing detergents used for protein extraction.
Although detergents are available from several commercial sources and used routinely in many research laboratories, the importance of detergent purity and stability is not widely appreciated. Detergents often contain trace impurities from their manufacture. Some of these impurities, especially peroxides that are found in most non-ionic detergents, will destroy protein activity. In addition, several types of detergents oxidize readily when exposed to the air or UV light, causing them to lose their properties and potency as solubilizing agents. Thermo Scientific Surfact-Amps Detergent Solutions provide high purity, low peroxide-containing detergents that are packaged under nitrogen gas in clear glass ampules. Providing convenience, quality, and consistency for all detergent applications.
However necessary and beneficial the use of detergent may have been for initial cell lysis or membrane protein extractions, subsequent applications or experiments with the extracted proteins may require removal of some or all of the detergent. For example, although many water-soluble proteins are functional in detergent-solubilized form, membrane proteins are often modified and inactivated by detergent solubilization as a result of native lipid interactions having been disrupted. In some such cases, membrane protein function is restored when they are reconstituted into bilayer membranes by replacement of detergent with phospholipids or other membrane-like lipid mixtures.
The function of an individual protein can be studied in isolation if it is first purified and then reconstituted into an artificial membrane (although recovery of native orientation in the membrane is a major challenge). Even where restoration of protein function is not an issue, detergent concentration may have to be decreased in a sample to make it compatible with protein assays or gel electrophoresis.
Detergent removal can be attempted in a number of ways. Dialysis is effective for the removal of detergents that have very high CMCs and/or small aggregation numbers, such the N-octyl glucosides. Detergents with low CMCs and large aggregation numbers cannot be dialyzed since most of the detergent molecules will be in micelles that are too large to diffuse through the pores of the dialysis membrane; only excess monomer can be dialyzed. Ion exchange chromatography using appropriate conditions to selectively bind and elute the proteins of interest is another effective way to remove detergent. Sucrose density gradient separation also can be used.
For Research Use Only. Not for use in diagnostic procedures.