- Mary Johnson Ph. D.mary at labome dot comSynatom Research, Princeton, New Jersey, United States
Detergents used in biomedical laboratories are mild surfactants (=surface acting agents), used for the disruption of cell membranes (cell lysis) and the release of intracellular materials in a soluble form. Detergents break the protein-protein, protein-lipid and lipid-lipid associations, denature proteins and other macromolecules, prevent unspecific binding in immunochemical assays and protein crystallization.
There are many types of detergents. New detergents, usually for specific applications, are continually being developed . This review the characteristics and applications of most common detergents.
|ionic||sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl|
|non-ionic||triton X-100, DDM, digitonin, tween 20, tween 80|
Detergents are organic compounds comprised of a hydrophobic hydrocarbon moiety and a hydrophilic charged headgroup (Fig. 1A). When dissolved in water at a given concentration and temperature, detergent molecules will form micelles, with the hydrophobic part in the interior of the micelle and the headgroup at the exterior (Fig. 1B). The hydrophobic core of the micelle binds to the hydrophobic regions of proteins. The number of detergent molecules in a micelle is the aggregation number, an important parameter used to assess membrane protein solubility . The length of the hydrophobic region is directly proportional to the degree of hydrophobicity and it is quite constant among detergents, while the charged headgroup is variable. Based on its characteristics, the common detergents are categorized into three types: ionic (anionic or cationic), zwitterionic and non-ionic. The minimal detergent concentration at which micelles are observed at a specific temperature is called Critical Micelar Concentration (CMC). At any concentrations lower than the CMC, only monomers are observed; at concentrations higher than CMC both micelles and monomers co-exist, along with other non-micellar phases that are not dissolved in water. Likewise, the lowest temperature at which micelles are formed is called Critical Micelar Temperature (CMT). Both temperature and concentration are important parameters of phase separation and solubility of a detergent. CMC is also affected by the degree of lipophilicity of the headgroup. Generally, a low lipophilic or lipophobic character results in high CMC.
Ionic detergents are comprised of a hydrophobic chain and a charged headgroup which can be either anion or cation. They generally have higher CMC value than nonionic detergents. These detergents are harsh.
Due to their charged headgroups, ionic detergents cannot be removed by ion exchange chromatography.
The anionic SDS is a very effective surfactant in solubilizing almost all proteins. It disrupts non-covalent bonds within and between proteins; thus it denatures them, resulting in the loss of their native conformation and function. SDS binds to a protein with a ratio 1.4:1 w/w (or one SDS anion per two amino acids) and therefore will mask the charge of the protein. SDS adds a negative charge to all proteins in the sample regardless of their isoelectric point (pI). That is the main reason for its wide use in SDS polyacrylamide gel electrophoresis (SDS-PAGE). Usually, for a complete cell lysis in the presence of SDS, a sample must be sonicated or passed through a needle (19G) several times to ensure DNA degradation. SDS should never be used when active proteins are required or when protein-protein interactions are studied. Additional precautions should be taken when ionic detergents are used because some of their properties may be altered in buffers with variable ionic strength (e.g. CMC falls down to 0.5 from 8 mM when the NaCl concentration increases from 0 to 500 mM). Furthermore, SDS precipitates at low temperatures, and this effect is enhanced in the presence of potassium salts. This phenomenon can be exploited to remove SDS from the protein sample .
Deoxycholate and cholate fall in this category even though they do not contain a charged headgroup. Their polar groups are distributed in different parts of the chains. They are used to solubilize membranes.
Sarkosyl, also known as sarcosyl or sodium lauroyl sarcosinate, is an anionic surfactant. It is amphiphilic due to the hydrophobic 14-carbon chain (lauroyl) and the hydrophilic carboxylate. The nitrogen atom in the amide linkage is not pH active and remains neutral regardless of pH. The carboxylate with a pKa value of 3.6 is negatively charged in any physiological solution. In addition, sarkosyl has a very high surface activity throughout a wide pH range, and its surface tension is about 3.4 × 10–2 to 2.9 × 10–2N/m.
Sarkosyl is prepared from lauroyl chloride and sarcosine in the presence of sodium hydroxide, and is purified by recrystallization from alcohol, or by acidification with mineral acid, separation of the free acid, and neutralization of the free acid. Sarkosyl reported in literatures is usually from Sigma. The most widely used commercial form of acyl sarcosine is a 30% aqueous solution.
Sarkosyl is widely used in cosmetic formulations as shampoos, body washes, facial washes, and surfactant-cleansing agents. Sarkosyl was used at concentrations of 2.78-12.9% in soaps, based on gas chromatographic analysis of the products . Sarkosyl is used in the metal finishing end processing industries for their crystal modifying, anti-rust, and anti-corrosion properties. Sarkosyl has been also used to improve wetting and penetration of topical pharmaceutical products. In food industry, sarkosyl is approved for use in processing, packaging, and transporting food for human consumption, and in adhesives used in food storage or transportation.
Sarkosyl is also widely utilized in laboratory experiments due to its good water solubility, high foam stability, and strong sorption capacity to proteins. Sarkosyl can inhibit the initiation of DNA transcription. Sarkosyl serves as a detergent to permeabilize cells and extract proteins in isolation and purification techniques such as western blot and indirect ELISA.
One major application of sarkosyl is for solubilizing and refolding proteins from inclusion bodies. Eukaryotic recombinant proteins overexpressed in Escherichia coli tend to form inclusion bodies. Sarkosyl can extract natively folded proteins from inclusion bodies. Usually an inclusion body pellet is solubilized in 0.3% sarkosyl. Incubation of inclusion bodies with 10% sarkosyl effectively solubilized > 95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ratio of Triton X-100 and CHAPS . Earlier work involved solubilizing inclusion bodies with denaturants such as urea or guanidinium hydrochloride and refolding by slow dilution. However, most of the solubilized proteins aggregate and precipitate upon removal of the strong detergents. Sarkosyl has been found to be an effective solubilizing agent that allows refolding at higher protein concentrations (as much as 10-fold higher when compared to using guanidinium hydrochloride  ). Moreover, sarkosyl allows refolding of solubilized protein with less aggregation than urea or guanidinium hydrochloride. Proteins in the soluble extract with sarkosyl can be stored at 4°C for a week before affinity purification. However, sarkosyl interferes with the subsequent chromatographic process and must be removed from the solution by dilution, or dialysis.
Nonionic detergents have uncharged and hydrophilic headgroups. They are considered mild surfactants as they break protein-lipid, lipid-lipid associations, but not protein-protein interactions, and most of them do not denature proteins. Therefore, proteins are solubilized and isolated in their native and active form, retaining their protein interactors. They are preferred for the isolation of membrane proteins.
Triton X-100 is a typical nonionic surfactant and the surfactant of choice for most immunoprecipitation experiments. All members of this family (Triton X100, Triton X114, Nonidet P40, Igepal® CA-630) are quite similar and differ only in their average number (n) of monomers per micelle (9.6, 8.0, 9.0, and 9.5, respectively) and in the size distribution of the PEG-based headgroup . Triton X100 derives from polyoxyethylene and contains an alkylphenyl hydrophobic group. The CMC value is low and therefore it can not be easily removed by dialysis. The cloud point is 64oC and at this temperature phase separation is observed.
The n-dodecyl-β-D-maltoside (DDM) is a glycosidic surfactant, increasingly used in hydrophobic and membrane protein isolation, when the protein activity needs to be preserved. It is proved to be more efficient than others, such as CHAPS, or NP-40 . The glycochain in its lipophilic site, its high CMC of 0.17 mM and the interface of the micelles create an aqueous-like microenvironment ideal for solubilizing and retaining the stability of membrane and hydrophobic proteins.
Digitonin, from Purple Foxglove (Digitalis purpurea), is used for the solubilization of eukaryotic plasma membranes. Sarkosyl and Triton X-100 solubilize bacterial inner, but not outer membranes.
Tween-20 and Tween-80 are polysorbate surfactants with a fatty acid ester moiety and a long polyoxyethylene chain. They have very low CMC and are generally gentle surfactants, they do not affect protein activity and they are effective in solubilization. They are not common ingredients of cell lysis buffers, they are routinely used as washing agents in immunoblotting and ELISA, in order to minimize unspecific binding of antibodies and to remove unbound moieties.
One common question regarding the Tween family detergents is the difference between Tween 20 and Tween 80, two most commonly used members. Tween 20 has lauric acid, while Tween 80 has oleic acid, see figure 3. Table 2 summarizes various aspects between them.
|Synonyms||Chemical Formula||Molecular Weight||Density (g/mL)||Appearance||Applications|
|Tween 20||polysorbate 20, polyoxyethylene sorbitan monolaurate, PEG (20) sorbitan monolaurate||C58H114O26||1228||1.1|
Clear, yellow to yellow-green viscous liquid
|a broad range of applications: as a blocking agent in PBS or TBS wash buffers for ELISA, Western blotting and other immunoassay methods; for lysing mammalian cells; and as a solubilizing agent for membrane proteins.|
|Tween 80||polysorbate 80, polyoxyethylene sorbitan monooleate, PEG (80) sorbitan monooleate||C64H124O26||1310||1.06-1.09||amber colored viscous liquid||as a stabilizing agent for proteins; used in tests for the identification of phenotype of some mycobacteria.|
Most non-ionic detergents interfere with ultra-violet (UV) spectrophotometry, especially Triton X100, as they contain a phenyl ring and they absorb UV light. Therefore, protein determination at 280 nm would be imprecise.
The headgroups of zwitterionic (or amphoteric) detergents’are hydrophilic and contain both positive and negative charges in equal numbers, resulting in zero net charge. They are more harsh surfactants than the nonionic ones. A typic zwitterionic detergent is 3- [(3-cholamidopropyl)dimethylammonio] -1-propanesulfonate, better known as CHAPS. Its high CMC (6 mM at room temperature) allows efficient removal by dialysis. It is very common in sample preparation at concentrations of 2-4% for isoelectric focusing and 2D electrophoresis. CHAPSO differs with CHAPS in that it contains a more polar headgroup, which makes it more soluble. Thus, CHAPSO is mainly used for solubilization of integral membrane proteins.
Chaotropic agents are similar substances to surfactants in that they break non-covalent interactions (hydrogen bonds, dipole-dipole interactions, hydrophobic interactions) facilitating protein denaturation, which in this case is usually reversible. Urea alone, or in combination with thiurea or with other detergents, is the most widely used chaotrope with applications in 2D-gel electrophoresis and in-solution enzymatic digestion of proteins for preparation during proteomic workflows. When using Urea, extra care must be taken to not heat the sample above 37oC as this will lead to carbamylation of proteins .
For membrane protein solubility, a detergent with high CMC should be chosen and the volume of the buffer would also be crucial as enough detergent should be present to solubilize all membrane proteins in the sample. According to Linke , at least on micelle is needed per one membrane protein in order to sufficiently mimic the lipid environment of a membrane (Fig. 1C-D). In principle, by changing the temperature and the concentration of salt in the buffer, an effective solubilization of membrane proteins can be achieved taking advantage of the phase separation. In this case, the membrane proteins surrounded by the micelles precipitate with the detergent and the soluble proteins remain in the supernatant. The temperature at which the detergent solution separates into two phases is called the cloud point. In addition to the temperature, the cloud point is affected by additives in the buffer such as glycerol or salts (e.g. Triton X114 has a cloud point of 23oC but in the presence of 20% glycerol, the cloud point declines to 4oC). This is very important since the stability of a protein is affected by high temperatures.
A good detergent should be able to lyse cells, solubilize proteins and be suitable for downstream application. In addition, the solublized protein in native or denatured form should be considered. There is no ideal detergent for all applications and even in the same application the result varies (Table 3). Therefore, trial and error is the best strategy and the use of a mixture of detergents should also be tested. In addition, fresh preparation of detergent working solution is usually the best practice to avoid hydrolysis and oxidation.
|Detergent||MW (Da) monomer||MW (Da) micelle||CMC (mM) 25oC||Aggregation No.||Cloud Point (oC)||Avg. Micellar Weight||Strenght||Dialyzable||Applications|
|SDS||289||18,000||7-10||62||>100||18,000||Harsh||Yes||Cell lysis, Electrophoresis, WB, hybridization|
|Triton X-100||625||90,000||0.2-0.9||100-155||65||80,000||Mild||No||Enzyme immunoassays, IP, Membrane solubilization|
|Tween-20||1228||0.06||76||mild||No||WB, ELISA, Enzyme immunoassays|
The downstream application controls not only the type of detergent but also its concentration, which usually should be lowered or completely removed. For such purposes, size exclusion chromatography or dialysis can be used but this requires that the micelles size is different compared to the protein of interest or small enough micelles (i.e high CMC) to pass the dialysis tubing . Other methods employ the use of non-polar beads or resins that bind detergents, cyclodextrin inclusion compounds , ion-exchange chromatography or protein precipitation. However, the end buffer after detergent removal should be also chosen with extreme care to avoid precipitation and protein aggregation.
Labome surveyed literature for the application of detergents. The following table lists the main suppliers, and number of the articles, indicating most of the detergents are supplied by Sigma Aldrich.
|Triton X-100||31||Thermo Fisher, Amresco, JT Baker, Packard, Sigma|
|Tween-20||19||Thermo Fisher, Amersham Pharmacia, Bio-Rad|
|SDS||13||Amresco, Bio-Rad, Q.BIOgene, Sigma|
|CHAPS||5||EMD Millipore/Merck/Calbiochem, Sigma, JT Baker|
|digitonin||6||EMD Millipore/Merck, Sigma, Wako|
Thermo Fisher Pierce Triton X-100 was used to lyse cell and tissue samples for western blots  . Amresco 30% TritonX-100 was used to homogenize the spinal cords of rats . JT Baker Triton X-100 was used to fix and permeabilize cells for visualizing the actin cytoskeleton . Packard Triton-X was used to dissolve primary antibodies for immunocytochemistry . Sigma Triton X-100 was used to permeabilize cells in immunocytochemistry         , or in blocking buffer for immunohistochemistry   , to solublize samples in western blots  , to lyze cells and tissue samples       , in PCR experiments , liposome fusion experiments , and in whole mount staining .
Tween-20 is commonly used in washing buffers, such as TBS-T, PBS-T, in various immuno assays.
Fisher 0.05% Tween was used in ELISA . Amersham Pharmacia Tween 20 was used in western blot . Bio-Rad Tween 20 was used in PBS for immunoblot analysis  and for washing tissue sections in immunohistochemistry . Sigma Tween-20 was used in washing blots  , , ,    , in ELISA  , in immunoprecipitation , in in situ hybridization , and in microfluidic array multiplex PCR  and others  .
Amresco SDS was used in SDS-PAGE . Bio-Rad sodium dodecyl sulfate was used to prepare radioimmunoprecipitation assay buffer . Q.BIOgene SDS was used to prepare buffer for homogenizing pellets in western blots . SIGMA SDS was used to prepare buffers for, among others, in vitro octanoylation assays, Laemmli sample buffer, 2D-DIGE experiments         . Sigma 10% SDS was used to homogenize the spinal cords of rats .
Calbiochem CHAPS (2% w/v) was used in order to identify circulating protein markers of ovarian cancer . Sigma CHAPS was used in buffers for purification of recombinant mouse proghrelin , immunoprecipitation assays , and tissue lysis . JT Baker CHAPS was used to lyse cells to study viral interaction with human ASF1 protein .
MERCK digitonin was used in immunocytochemistry experiment to study therole of PI4P in plasma membrane identity . Sigma digitonin was used to permeabilize cells to study the epithelial and mesenchymal cell responses towards Escherichia coli Shiga toxin 1 , to study autophagy , and to prepare extraction buffer for the study of TNFalpha effect on macrophages . Wako digitonin was used to lyze cells  and perform immunoprecipitaion experiments .
For protein purifications, Anatrace n-decyl-beta-D-maltopyranoside was used for protein purification  and at 5 mM , Glycon n-octyl-beta-D-glucopyranoside  and beta-dodecyl-maltoside , Affymetrix octyl glucose neopentyl glycol (OGNPG) at 1 % , and Sigma cholesteryl hemisuccinate at 0.1% (w/v) .
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