HeLa Cells Explained
An Exclusive Look at Real HeLa Cells
An Exclusive Look at Real HeLa Cells
Immortal Cells Turn 96
The immortal cells of Henrietta Lacks - Robin Bulleri
Winfrey, Byrne and Goldsberry on "The Immortal Life of Henrietta Lacks"
HeLa is an immortal cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, from Henrietta Lacks, a 31-year-old African-American mother of five, who died of cancer on October 4, 1951. Wikipedia
Cell Lysis
Super-resolution microscopy of bursting bacteria
OpenCEL Cell Lysis Technology for Biosolids Reduction HD HD
Protozoa death: osmotic shock cell lysis
- the disintegration of a cell by rupture of the cell wall or membrane.
Lysis
Lysis (/ˈlaɪsɪs/ LY-sis; Greek λύσις lýsis, "a loosing" from λύειν lýein, "to unbind") is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" /ˈlɪtɪk/ LIT-ək) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.
Many species of bacteria are subject to lysis by the enzyme lysozyme, found in animal saliva, egg white, and other secretions.[1] Phage lytic enzymes (lysins) produced during bacteriophage infection are responsible for the ability of these viruses to lyse bacterial cells.[2] Penicillin and related β-lactam antibiotics cause the death of bacteria through enzyme-mediated lysis that occurs after the drug causes the bacterium to form a defective cell wall.[3] If the cell wall is completely lost and the penicillin was used on gram-positive bacteria, then the bacterium is referred to as a protoplast, but if penicillin was used on gram-negative bacteria, then it is called a spheroplast.
Immune response[edit]
Erythrocytes' hemoglobin release free radicals in response to pathogens when lysed by them. This can damage the pathogens.[6][7]
Cell lysis
Cell lysis is a common outcome of viral infection. It consists of a disruption of cellular membranes, leading to cell death and the release of cytoplasmic compounds in the extracellular space.
Lysis is actively induced by many viruses, because cells seldom trigger lysis on their own. Indeed eukaryotic cells rather tend to trigger apoptosis when attacked by viruses.
Lytic replication: Most non-enveloped virus, and few enveloped viruses require cell lysis in order to release new virions from the infected cell. Cell lysis is actively induced by viruses using various mechanisms:
Viroporins: Some eukaryotic lytic viruses like the Adenoviridae , and Picornaviridae encode viroporins in the late phase of infection in order to disrupt the cell membrane.
Lytic phospholipids: Phycodnaviridae may induce the synthesis of lytic phospholipids 
.
Bacteria lysis: All bacterial viruses are lytic, except filamentous phages and plasmaviridae. Most bacteriophages with a complex capsid use the endolysins/holins/spanins lysis mechanism. Microviridae and Leviviridae inhibit cell wall biosynthesis and induce cytolysis.
Occasional lysis: Many viruses can induce cell lysis under special circumstance.
Occlusion body: In the late phase of host infection some viruses induce the formation of a crystalline protein matrix that ends up with cell lysis. The viruses trapped in the occlusion body are often involved in a host-to-host infection. This strategy is used by Baculoviridae , insects infecting Iridoviridae , Cypovirus and insects infecting Poxviridae .
Immune response: In vertebrate hosts, infected cell lysis can be induced by natural killer cells or cytotoxic T cells responding to the infection.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323333/
Thymol and carvacrol induce autolysis, stress, growth inhibition and reduce the biofilm formation by Streptococcus mutans
Introduction
The oral cavity is a complex microbial environment hosting around 600 different bacterial species and many of these bacteria are now being associated with oral diseases (Dewhirst et al. 2010; Moore and Moore 1994; Wade 2013). Dental diseases incur substantial economic losses globally accounting to 298 billion US dollar per year which is 4.6% of the total global health expenditure (Listl et al. 2015). The evidence is also now available that the recurring infections of these oral microorganisms result in a number of systemic diseases further adding to the economic losses and loss of life (Khan et al. 2015; Li et al. 2000). One of the most important etiological agents of dental caries is Streptococcus mutans (Loesche 1986). In addition to being classically associated with dental caries, this bacterium also causes other systemic diseases such as ulcerative colitis, endocarditis and septicemia (Kojima et al. 2012; Nobbs 2016; Robbins et al. 1977; Tunkel and Sepkowitz 2002). The biofilm formation by S. mutans and its ability to promote the biofilm formation by other oral bacteria also makes it more difficult to treat the infections of S. mutans (Ahn et al. 2008; Klein et al. 2015; Krzyściak et al. 2014). Therefore, for a good oral hygiene, it is very important to control the growth of S. mutans in the oral cavity. Furthermore, the unwarranted and overuse of the antibiotics in dentistry has resulted in the drug resistance among commensal as well as pathogenic bacteria of the oral cavity including S. mutans (Leistevuo et al. 2000; Sweeney et al. 2004). Therefore, alternatives to conventional antimicrobial agents are highly and urgently required. Among, the alternative antimicrobials are engineered nanomaterials, plant-based materials, bacteriocins and phage-based therapies (Allen et al. 2014; Khan et al. 2016b, c; Rose et al. 2014).
Plant based materials, especially from edible plants, are an effective alternative antimicrobials especially those classified as GRAS (generally recognized as safe) (Burdock and Carabin 2004). Two such commonly used edible plants in traditional medicine since ancient times are Origanum and Thymus (Craig 1999; Nostro and Papalia 2012). Essential oils from these plants have been shown to exhibit significant antimicrobial activities and their antimicrobial activity against food-borne pathogens have been already tested (Chorianopoulos et al. 2004). The major components in the essential oils of Origanum and Thymus are the phenolic monoterpene carvacrol and its isomeric form Thymol (Chorianopoulos et al. 2004; Fachini-Queiroz et al. 2012). Both Carvacrol and Thymol have been classified as GRAS and their use in food has been approved by European Parliament and Council (Hyldgaard et al. 2012). The antimicrobial activity of thymol and carvacrol have been reported against foodborne pathogens such as Clostridium perfringens, Escherichia coli O157:H7 and Listeria innocua (Du et al. 2015; Guarda et al. 2011). The mechanism behind this antibacterial activity includes permeabilization and depolarization of the cytoplasmic membrane, by reducing the pH gradient across the cytoplasmic membrane. This reduction in pH gradient also adversely affects the proton motive force leading to the depletion of the intracellular ATP subsequently leading to cell death (Ultee et al. 2002; Xu et al. 2008). It is also concluded in the same study that the phenolic hydroxyl group of thymol and carvacrol plays an important role in its antimicrobial activity (Ultee et al. 2002).
In this paper, we report a detailed study on antimicrobial and antibiofilm activities of two natural compounds M-1 and M-2 isolated from O. vulgare L. Moreover, comparison of antimicrobial and antibiofilm activities of these two compounds with chlorhexidine digluconate a commercially used antiplaque compound and clove oil is also discussed in detail in this study.
Antimicrobial activity of test compounds by microdilution method
Antimicrobial activity was determined by using microdilution method. S. mutans was grown with 1000, 500, 250, 125, 62.5, 31, and 15 and µg/ml of clove oil, M-1, and M-2. Highest antimicrobial activity against S. mutans ATCC 25175 was observed with thymol (M-2), where almost no growth was observed with 125 µg/ml of M-2 or thymol. While for M-1or carvacrol complete inhibition of growth was observed with 250 µg/ml. Clove oil appeared to be the least effective in inhibiting the growth of S. mutans wherein even with 1000 µg/ml of concentration complete inhibition of the growth was not observed. Chlorhexidine digluconate which was used as a positive control inhibited the growth of S. mutans completely at a concentration of 10 µg/ml and was found to be almost twenty times more effective than carvacrol and thymol.
Change in bacterial viability as determined by dead/live staining
Live and dead staining was performed after S. mutans was grown with 25 µg/ml of carvacrol (M-1), thymol (M-2) and clove oil. While cells were also grown with 2.5 µg/ml of chlorhexidine digluconate as a positive control. Thymol (M-2), was found to be the most effective compound which resulted in the death of 42 ± 4% of the cells (Fig. 3b). Carvacrol (M-1) and clove oil resulted in the death of 39 ± 4 and 5 ± 2% of the cells at a concentration of 25 µg/ml. While the positive control chlorhexidine digluconate resulted in the death of 37 ± 3% of the cells at a concentration of 2.5 µg/ml. Figure 4 shows the BacLight staining for live and dead cells after treatment with test compounds. It is clear from the figure that in untreated controls cells appeared more dispersed and were found to be arranged in clusters. Whereas the cells treated with thymol, carvacrol and chlorhexidine appeared in short chains and show significantly low density.
The change in the viability of S. mutans as determined by Live/dead staining when untreated (a, CONT), treated with 25 µg/ml of clove oil (b, CO), carvacrol (d, M-1) and thymol (e, M-2) and 2.5 µg/ml of chlorhexidine digluconate (c, CHD). Orange or red cells represent dead cells while green cells represent live cells (refer to Additional file 1 for raw data)
For qualitative assessment, biofilms of S. mutans grown in 48 well plates in the presence of test compounds or without test compounds were observed under a scanning electron microscope as described in materials and methods. Untreated controls show uniform well grown and densely arranged bunches of cells (Fig. 6A), almost similar pattern was observed for the cells treated with clove oil (data not shown). On the contrary, the cells treated with thymol and carvacrol appeared as short chains with considerably reduced cell density. The cells grown with chlorhexidine show the least density of the cells. Moreover, cells treated with thymol, carvacrol, and chlorhexidine digluconate appeared lysed and deformed and are marked with arrows in Fig. 6 B, C.
Real-time PCR analysis of the genes involved in cell death, stress and biofilm formation
Change in the expression of six genes namely autolysin like genes AtlE (N-acetylmuramoyl-l-alanine amidase type), and AtlA like genes, Polyribonucleotide nucleotidyl transferase as a marker of general stress (PnpA), superoxide dismutase gene (SOD), ymcA and gtfB genes was studied to check the cell death, general stress and biofilm formation activities. Since it was found that two genes PnpA like and AtlA like genes were not sufficiently amplified in PCR assay using genomic DNA from S. mutans as template, these two genes were not used in RT-PCR studies. Furthermore, the sequence of the PCR products amplified using the PCR primers confirms that these primers are specifically binding to the target genes (Additional file 1).
RT-PCR analysis shows an increase of 2.4, 2.2 and 1.3 folds in the expression of Autolysin gene AtlE when S. mutans was grown with 15 µg/ml of thymol (M-2), carvacrol (M-1) and clove oil, respectively (Fig. 7). This increase in the expression of AtlE gene suggests that exposure to thymol and carvacrol induces apoptosis-like activity. Similarly, when S. mutans was grown with the same concentration of thymol (M-2), carvacrol (M-1) and clove oil, the expression of ymcA gene was upregulated by 2.1, 1.7 and 1.2 folds, respectively. The expression level of sodA genes also increased by 1.43, 1.32, and 1.1 when grown with 15 µg/ml of thymol (M-2), carvacrol (M-1) and clove oil, respectively. An increase in the expression of these genes shows that exposure to thymol and carvacrol results in an increase in oxidative stress and general stress in S. mutans. On the contrary the expression level of gtfB decreased by 0.3, 0.3 and 0.1 folds, when grown with thymol (M-2), carvacrol (M-1) and clove oil, respectively. This decrease in gtfB gene expression suggests the inhibition of biofilm formation which was also observed in other experiments. Similar, trend was observed, with the positive control chlorhexidine digluconate wherein a 2.9, 3.3 and 2.1 folds increase in the expression of ymcA, autolysin AtlE, and sodA genes, respectively was observed at a concentration of 1 µg/ml. While the exposure to chlorhexidine digluconate resulted in 0.4 folds decrease in the expression of the gtfB gene.
Discussion
Essential oils from edible plants especially those classified as GRAS are one of the safe alternatives to traditional antibiotics (Kalemba and Kunicka 2003). But the commercialization of these plant-based products is often hampered by the inability to purify bioactive compounds from these plants. Two such compounds, thymol, and carvacrol are the main constituents of O. vulgare L., both compounds are known to exhibit antimicrobial activity against a number of pathogens (Chorianopoulos et al. 2004; Thosar et al. 2013; Ultee et al. 2002). Interestingly, an in vitro study has demonstrated that thymol, and carvacrol show selectively higher antimicrobial activity against the tested pathogenic bacteria (Escherichia coli, Clostridium perfringens, and Salmonella) than the beneficial probiotic bacteria Lactobacillus (Du et al. 2015). When thymol and carvacrol were tested in vivo for their efficacy to inhibit C. perfringens in broilers it was found that although the population of the pathogen was not reduced significantly but the treatment alleviated intestinal lesions caused by these pathogens (Du et al. 2015). The same group (Du et al. 2016) in their in vivo study on broilers has further demonstrated many beneficial effects of thymol and carvacrol including an increase in feed conversion efficiency, increase in immunity against virus and tumor. It has been reviewed earlier also that herbs that serve as a primary source of these essential oils exhibit anticancer activities (Craig 1999). Hence, thymol and carvacrol in addition to possessing desired antimicrobial activity also have known health benefits making these essential oils a promising alternative antimicrobial agent.
Dental caries and periodontal diseases are most prevalent microbial diseases and S. mutans is one of the most important bacterium involved in dental caries (Loesche 1986; Selwitz et al. 2007). To keep a good oral hygiene, it is very important to check the growth of S. mutans. However, over and unwarranted use of antibiotics has also resulted in the development of antibiotic resistance in these pathogens (Leistevuo et al. 2000). Since, thymol and carvacrol exhibit good antimicrobial activity these essential oils were purified from O. vulgare L. and their antimicrobial activity against S. mutans was determined. Furthermore, the antimicrobial activity of thymol and carvacrol was compared with clove oil, a traditionally used essential oil in dentistry, and with chlorhexidine digluconate a widely used compound in mouthwashes (McBain et al. 2003; Thosar et al. 2013). To our knowledge, this is the first such detailed report on the antimicrobial and antibiofilm activity of thymol and carvacrol against S. mutans.
IC50 values of thymol, carvacrol, clove oil and chlorhexidine digluconate were found to be 54, 65, 306 and 4.9 µg/ml, respectively in this study. These results were further confirmed by MTT assay wherein 200 µg/ml of thymol and carvacrol reduced the cell viability by 87 ± 6 and 74 ± 8%, respectively. While, chlorhexidine digluconate resulted in the death of 37 ± 3% of the cells at a very low concentration of 2.5 µg/ml. In earlier studies, the essential oils from thyme and origanum exhibited high MIC values in a range of 256–512 μg/ml against group A Streptococci (GAS) (Magi et al. 2015). While in the same study commercially available carvacrol exhibited a MIC value in the range of 64–256 µg/ml against GAS (Magi et al. 2015). Decontamination of lettuce using a solution of carvacrol and thymol was also reported since these essential oils are edible (Bagamboula et al. 2004). In another study, the antimicrobial activity of microencapsulated carvacrol and thymol was determined against many microorganisms including foodborne pathogens Escherichia coli O157:H7, Staphylococcus aureus, and Listeria innocua. The MIC of the microencapsulated carvacrol and thymol against these organisms were in the range of 225–375 ppm (Guarda et al. 2011). The positive control chlorhexidine digluconate suppressed the growth and viability of S. mutans at much lower concentrations as found in earlier studies also (McBain et al. 2003). While, the MIC values of eugenol a main constiuent of clove oil against S. mutans were reported to be 100 µg/ml (Freires et al. 2015).
As far as the mechanism of the antimicrobial activity is concerned, it is suggested that these compounds are involved in the permeabilization and depolarization of the cytoplasmic membrane. Which results in the reduction of the pH gradient across the cytoplasmic membrane. This lowering of pH gradient leads to the disturbance in proton motive force subsequently leading to the depletion of intracellular ATP level and cell death (Ultee et al. 2002). The increase in the number of dead cells following the treatment with thymol and carvacrol was also observed in this study (Figs. 3b, b,4)4) through propidium iodide staining a dye that can only penetrate into the cells having compromised cell wall. Deformed and lysed cells as observed under scanning electron microscope further confirms that thymol and carvacrol results in the lysis of the cells. The overexpression of autolysin genes involved in the restructuring of the cell wall further confirms these findings. Our results also show the over expression sodA and ymcA genes, suggesting that treatment with thymol and carvacrol induces general and oxidative stress in the cells. Chlorhexidine digluconate also exhibit its antimicrobial activity primarily through membrane disruption (McBain et al. 2003). Inhibition of glycosidic and proteolytic enzymes by chlorhexidine digluconate is also reported (Hastings 2000). Although carvacrol and thymol also disrupt the cell membrane but these essential oils are not water-soluble which may adversely affect their penetration into the bacterial cells resulting in lower activity. This argument is also supported by the findings that nano form of thymol exhibit better antimicrobial activity than the native form because of the improved dispersibility (Shah et al. 2012).
Another important trait of pathogenicity is biofilm formation. Biofilms exhibit greater resistance to antimicrobial agents and are difficult to treat (Ahn et al. 2008; Curtis et al. 2011). Therefore, the antibiofilm activities of carvacrol (M-1) and thymol (M-2) against S. mutans was determined. The results of crystal violet assay and the qualitative examination of the biofilms under SEM (Fig. 6) suggest that the two compounds significantly reduce the biofilm formation by S. mutans. In gene expression studies also the downregulation of glycosyl transferase B (gtfB) gene suggests the inhibition of biofilm formation. Previously the antibiofilm activity of thymol and carvacrol against Pseudomonas aeruginosa has been reported (Ceylan and Ugur 2015). Chlorhexdine digluconate also reduced the biofilm formation at a concentration of 10 µg/ml. Inhibition of biofilm formation by chlorhexidine has been reported in in vivo studies also (Bailón-Sánchez et al. 2014). As far as the antimicrobial and antibiofilm activities of clove oil are concerned clove oil was found to be least effective.
Results presented in this study strongly suggest that thymol and carvacrol exhibit significant antimicrobial and antibiofilm activities against S. mutans. Since these compounds are derived from edible plants classified as GRAS, these essential oils can be used in mouthwashes or toothpastes for controlling oral bacteria and for maintaining good oral hygiene. Furthermore, unlike chlorhexidine digluconate, thymol and carvacrol have some health benefits in addition to the desired antimicrobial activity. Although, chlorhexidine digluconate is widely used and do not have any known health hazard but its effect on taste buds is well known (Helms et al. 1995).
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Surfactant-induced cell toxicity and cell lysis. A study using B16 melanoma cells
- PMID: 2403386
- DOI: 10.1016/0006-2952(90)90399-6
Abstract
The effects of a variety of detergents (non-ionic, ionic and bile derivatives) on B16 melanoma cells have been examined. Two main effects can be clearly differentiated: loss of cell viability and cell lysis. Under our conditions, cell-surfactant interaction is highly dependent on the nature of the amphiphile (more specifically, on its critical micellar concentration). Loss of cell viability occurs at surfactant concentrations below the critical micellar concentration, i.e. the incorporation of detergent monomers into the cell membranes is enough to impair their barrier function, so that Trypan Blue is no longer actively secreted outside the cell. On the other hand, cell lysis only occurs at or near the critical micellar concentration of the detergent, i.e. when the bilayer-micelle transition may take place. Comparative studies using B16 cells and phospholipid vesicles indicate that the amount of detergent required to induce cell lysis is the same that produces disruption of the lipid bilayer. Thus, our results suggest that membranes are the primary target for the toxicologic effects of surfactants on cells. Moreover, they provide a rationale for the interpretation of other studies in this field: previous results from different laboratories are shown to fit very well our data.
Beyond de-foaming: the effects of antifoams on bioprocess productivity
Conclusion
The biological effects of antifoams are poorly understood and this is in part due to the range of types available and the lack of information regarding their compositions being available from the manufacturers. Antifoams have commonly been added to bioprocesses without full knowledge of their possible effects, but as an additive, these effects should be assessed. Published studies have demonstrated that each antifoam not only destroys foam with a range of effectiveness, but may also affect the cells and the proteins themselves. The concentration and type of antifoam required to alleviate foam should therefore be balanced with the possible effects it could have upon the process. Consequently, screening for optimum conditions is required. Our study and that of Koch et al demonstrated that higher concentrations of antifoam than would normally be used can benefit the process, however it has also been suggested that antifoams could damage fermentation equipment[13], and they are known to foul membranes in downstream processing[53]; therefore consideration of the whole process must be taken. In summary, these investigations have illustrated that antifoams could increase the productivity of a process or hinder it. It is not likely that the precise mechanisms of antifoams action will be easily understood, especially as a combination of factors may have led to the effects upon protein yields. For these reasons, it is important to thoroughly evaluate the effects of antifoam addition to fermentation cultures on both a small and large scale on a case-by-case basis.
Can anyone recommend a natural foaming agent as an alternative ...
Can anyone recommend a natural foaming agent as an alternative to SLS?
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https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/detergents-cell-lysis-protein-extraction.html
Detergents for Cell Lysis and Protein Extraction
Properties and types of detergents
Structure of surfactants. Generalized structure of a single detergent molecule (top) and the complete structure of CHAPS (bottom), an example of a zwitterionic detergent.
Chemical definition of detergent
Detergents are amphipathic molecules, meaning they contain both a nonpolar "tail" having aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for broad classification of detergents; they may be ionic (charged, either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero).
Detergents in solution
Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their nonpolar 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 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 nonpolar ends are sequestered inward, avoiding exposure to water, and the polar ends are oriented outward in contact with the water.
Idealized structure of a detergent micelle.
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 nonionic detergents decreases with increasing temperature, while the CMC of ionic detergents decreases with 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 nonionic detergents such as Triton X-100, bile salts such as cholate, and zwitterionic detergents such as CHAPS.
Properties of common detergents.
| Detergent | Type | Agg.#‡ | MW mono (micelle) | CMC mM (%w/v) | Cloud point °C | Dialyzable |
|---|---|---|---|---|---|---|
| Thermo Scientific Triton X-100 | Nonionic | 140 | 647 (90K) | 0.24 (0.0155) | 64 | No |
| Thermo Scientific Triton X-114 | Nonionic | – | 537 ( – ) | 0.21 (0.0113) | 23 | No |
| NP-40 | Nonionic | 149 | 617 (90K) | 0.29 (0.0179) | 80 | No |
| Thermo Scientific Brij-35 | Nonionic | 40 | 1225 (49K) | 0.09 (0.0110) | >100 | No |
| Thermo Scientific Brij-58 | Nonionic | 70 | 1120 (82K) | 0.08 (0.0086) | >100 | No |
| Thermo Scientific Tween 20 | Nonionic | – | 1228 ( – ) | 0.06 (0.0074) | 95 | No |
| Thermo Scientific Tween 80 | Nonionic | 60 | 1310 (76K) | 0.01 (0.0016) | – | No |
| Octyl glucoside | Nonionic | 27 | 292 (8K) | 23-24 (~0.70) | >100 | Yes |
| Octyl thioglucoside | Nonionic | – | 308 ( – ) | 9 (0.2772) | >100 | Yes |
| SDS | 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.
Purified detergent solutions
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 nonionic 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. We offer several high purity, low peroxide–containing detergents that are packaged under nitrogen gas in clear glass ampules. These Thermo Scientific Surfact-Amps Detergent Solutions provide unsurpassed convenience, quality and consistency for all detergent applications. A sampler kit includes 10 different purified detergents (seven in the Surfact-Amps format and three in solid form).
Structure of cell membranes
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 nonpolar 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.
Membrane disruption, protein binding and solubilization
Generally, moderate concentrations of mild (i.e., nonionic) 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 nonpolar 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 1 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.
Detergent removal methods
Removal of detergent from solubilized proteins
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 ways. Dialysis is effective for 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.
Watch this video to learn more about protein dialysis
Learn more
- Protein Cleanup Technical Handbook
- Tech Tip #13: Remove detergents from protein samples
Recommended reading
- Walker JM (2009) The Protein Protocols Handbook. Third Edition. New York (NY): Springer-Verlag New York, LLC.
For Research Use Only. Not for use in diagnostic procedures.
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Cell Lysis and Fractionation
Our tissue lysis, cell lysis, and cell fractionation products are optimized for sample type. The lysates or fractions collected are compatible with a wide range of downstream applications. Features include:
- High protein yields from cells or tissues
- Gentle formulations that preserve protein activity for downstream assays
- Directly compatible with protein assays, immunoprecipitation, immunoassays, western blotting, EMSA, and enzyme assays
- Validated using multiple tissue types and cell lines
- Eliminates the need for mechanical cell disruption
Featured cell lysis and fractionation categories
High-purity, low-peroxide, surfactant solutions and detergent solids for use in cell lysis reagent formulation, protein solubilization procedures, and as wash buffers for ELISA and other protein research methods.
Rapid, high-purity isolations from crude fractions or fractions of common densities—can be used to isolate a number of different organelles and organelle-specific proteins in small volumes, and can replace time-consuming and labor intensive density gradient centrifugations.
Gentle whole cell lysis buffers for high-yield protein extraction and solubilization from E. coli and other bacteria, cultured mammalian cells and tissues, insect cells, yeast cultures, and flowering plants.
Reagent-based kits optimized for stepwise separation, enrichment, and extraction of proteins from different cell fractions; isolate cytoplasmic, membrane, nuclear, chromatin-bound, and cytoskeletal proteins for downstream analysis.
Optimized reagent kits for subcellular fractionation of proteins and isolation of organelles such as nuclei, mitochondria, peroxisomes, and lysosomes by Dounce-homogenization and density-gradient separation.
Ready-to-use, inhibitor cocktail solutions and tablets, as well as individual purified formulations of protease inhibitors and phosphatase inhibitors to protect proteins from degradation during cell lysis and extraction.
Detergents for Cell Lysis and Protein Extraction
Properties and types of detergents
Structure of surfactants. Generalized structure of a single detergent molecule (top) and the complete structure of CHAPS (bottom), an example of a zwitterionic detergent.
Chemical definition of detergent
Detergents are amphipathic molecules, meaning they contain both a nonpolar "tail" having aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for broad classification of detergents; they may be ionic (charged, either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero).
Detergents in solution
Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their nonpolar 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 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 nonpolar ends are sequestered inward, avoiding exposure to water, and the polar ends are oriented outward in contact with the water.
Idealized structure of a detergent micelle.
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 nonionic detergents decreases with increasing temperature, while the CMC of ionic detergents decreases with 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 nonionic detergents such as Triton X-100, bile salts such as cholate, and zwitterionic detergents such as CHAPS.
Properties of common detergents.
| Detergent | Type | Agg.#‡ | MW mono (micelle) | CMC mM (%w/v) | Cloud point °C | Dialyzable |
|---|---|---|---|---|---|---|
| Thermo Scientific Triton X-100 | Nonionic | 140 | 647 (90K) | 0.24 (0.0155) | 64 | No |
| Thermo Scientific Triton X-114 | Nonionic | – | 537 ( – ) | 0.21 (0.0113) | 23 | No |
| NP-40 | Nonionic | 149 | 617 (90K) | 0.29 (0.0179) | 80 | No |
| Thermo Scientific Brij-35 | Nonionic | 40 | 1225 (49K) | 0.09 (0.0110) | >100 | No |
| Thermo Scientific Brij-58 | Nonionic | 70 | 1120 (82K) | 0.08 (0.0086) | >100 | No |
| Thermo Scientific Tween 20 | Nonionic | – | 1228 ( – ) | 0.06 (0.0074) | 95 | No |
| Thermo Scientific Tween 80 | Nonionic | 60 | 1310 (76K) | 0.01 (0.0016) | – | No |
| Octyl glucoside | Nonionic | 27 | 292 (8K) | 23-24 (~0.70) | >100 | Yes |
| Octyl thioglucoside | Nonionic | – | 308 ( – ) | 9 (0.2772) | >100 | Yes |
| SDS | 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.
Purified detergent solutions
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 nonionic 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. We offer several high purity, low peroxide–containing detergents that are packaged under nitrogen gas in clear glass ampules. These Thermo Scientific Surfact-Amps Detergent Solutions provide unsurpassed convenience, quality and consistency for all detergent applications. A sampler kit includes 10 different purified detergents (seven in the Surfact-Amps format and three in solid form).
Structure of cell membranes
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 nonpolar 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.
Membrane disruption, protein binding and solubilization
Generally, moderate concentrations of mild (i.e., nonionic) 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 nonpolar 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 1 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.
Detergent removal methods
Removal of detergent from solubilized proteins
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 ways. Dialysis is effective for 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.
Watch this video to learn more about protein dialysis
Learn more
- Protein Cleanup Technical Handbook
- Tech Tip #13: Remove detergents from protein samples
Recommended reading
- Walker JM (2009) The Protein Protocols Handbook. Third Edition. New York (NY): Springer-Verlag New York, LLC.
For Research Use Only. Not for use in diagnostic procedures.
OpenCEL Cell Lysis Technology for Biosolids Reduction HD HD
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