Behind the Blot: Everything You Need to Know About Tween 20

Tween‑20 (polysorbate 20) was first developed in the 1940s as part of the “Span and Tween” series of surfactants by Imperial Chemical Industries. Originally invented for industrial use as emulsifiers (notably in food and cosmetics), these non-ionic detergents quickly became staple ingredients in various applications. Tween is actually a trademark registered by ICI Americas, Inc., and the number “20” refers to the total ethylene oxide units in the molecule. By the mid-20th century, Tween‑20 began appearing in scientific protocols – for example, early immunoassay techniques in the 1970s incorporated Tween‑20 in wash buffers to reduce background noise. As enzyme-linked immunosorbent assays (ELISAs) were pioneered in 1971, researchers adopted 0.05% Tween‑20 in the wash steps to improve specificity. One of the earliest studies to apply Tween-20 in immunoassays was published in 1985 by Spinola and Cannon. In their Western blot experiments, they demonstrated that Tween-20 effectively blocked non-specific sites on membranes, performing as well as or better than traditional blocking agents. This finding paved the way for the widespread use of Tween-20 in modern immunodetection techniques. It has since transitioned from being purely an industrial emulsifier to an indispensable reagent in life science research. Today, Tween‑20 remains ubiquitous in labs worldwide and is even formulated into many commercial kits and automated staining systems, underscoring its enduring role in modern science.

Figure: Structural representation of Polysorbate 20 (Tween‑20), showing the sorbitan (sorbitol-derived) backbone with polyoxyethylene chains (totaling ~20 ethylene oxide units across four arms) and a lauric acid fatty acyl ester tail. (image from Wikipedia)

Chemically, polysorbate 20 is defined as polyoxyethylene (20) sorbitan monolaurate. In simpler terms, it is derived from a sorbitan ring (which comes from sorbitol, a sugar alcohol) that has been esterified with a fatty acid (lauric acid, a 12-carbon saturated acid), and then ethoxylated with an average of 20 ethylene oxide units (polyethylene glycol chains). This gives Tween‑20 a molecular formula of approximately C58H114O26 and an average molar mass around 1,225–1,280 Da. Importantly, Tween‑20 is not a single uniform molecule but a mixture of species: the ethoxylation process yields a distribution of chain lengths (totaling about 20 EO units on average across 4 chains). Likewise, while laurate is the primary fatty acid, commercial Tween‑20 often contains small fractions of other fatty acids (myristate, palmitate, stearate) as impurities or byproducts (lauric acid typically ≥40% of the mixture).

Tween 20 is a non-ionic surfactant, which means it has hydrophilic polyethylene glycol groups and a hydrophobic fatty tail, yet carries no net charge. It is supplied as a clear, viscous, pale yellow liquid at room temperature. Key properties include a hydrophile–lipophile balance (HLB) of ~16.7 (highly hydrophilic) and a critical micelle concentration (CMC) on the order of 0.05–0.07 mM (around 60 mg/L at 20–25 °C). In practical terms, this means Tween 20 is readily soluble in water (forming micelles at very low concentrations) and in polar organic solvents like lower alcohols and dioxane, but is insoluble in hydrocarbons and natural oils. It is relatively stable and inert in neutral aqueous conditions, being mild and non-denaturing to proteins. However, it can be an irritant in pure form and will decompose if exposed to extreme conditions (see “Limitations” below). Its non-ionic nature ensures that it does not interfere with the ionic strength or pH of buffers, making it compatible with most biochemical reagents.

Tween 20 is most often encountered in the laboratory as a component of buffer solutions at low percentages. It is typically sold as a stock solution (for example, 10% or 20% v/v) and then diluted into working solutions. Table 1 below summarizes some common formulations containing Tween 20 and their typical concentrations:

Table 1: Typical buffer formulations containing Tween‑20 and their concentrations. (PBS = phosphate-buffered saline; TBS = tris-buffered saline; BSA = bovine serum albumin; IF = immunofluorescence; IHC = immunohistochemistry.)

In practice, concentrations of Tween‑20 in working solutions are kept quite low (often 0.05–0.1% v/v, equivalent to 0.5–1 mL per liter) because even tiny amounts are effective at preventing non-specific adsorption of proteins. For instance, a classic ELISA wash buffer might be PBS + 0.05% Tween‑20, and Western blot wash buffers are typically TBS + 0.05% or 0.1% Tween‑20 (TBST). In immunostaining protocols (IHC/IF), 0.1% is a common choice for both washes and antibody diluents. Higher concentrations (0.5–2%) are used only in special cases such as cell lysis or membrane protein extraction where a stronger detergent effect is needed. Notably, stock solutions of Tween‑20 can be quite viscous, so labs often prepare a 10% solution for easier pipetting; many vendors sell 10% Tween‑20 ready-to-use for buffer preparation.

ELISA (Enzyme-Linked Immunosorbent Assay)

In ELISA procedures, Tween‑20 plays a critical role in wash buffers and sometimes in blocking solutions. After coating an ELISA plate with antigen or antibody and performing binding steps, the plate must be washed thoroughly to remove unbound enzyme conjugates and reduce background. Tween‑20 (0.05–0.1%) in PBS or TBS is routinely added to these wash buffers to prevent nonspecific protein–surface interactions. The detergent helps to “wet” the plastic wells uniformly and dislodge loosely bound proteins without disrupting specific antibody-antigen binding. This yields a cleaner signal and higher signal-to-noise ratio, particularly when assays are developed using well-defined antigens generated through custom peptide synthesis. Indeed, it is standard practice that “a detergent such as 0.05% Tween-20” be included in ELISA wash steps to help remove nonspecifically bound material.

Tween‑20 may also be present in the blocking buffer or antibody diluent in ELISAs. For example, a blocking buffer of 1–5% BSA or milk in PBS might include ~0.05% Tween‑20 to further reduce background binding on the polystyrene well surface. Including Tween‑20 along with the blocking protein minimizes hydrophobic interactions that could cause proteins to stick nonspecifically. The outcome is decreased background staining and more reliable, low-noise readings. Overall, the addition of Tween‑20 is one reason ELISA can achieve high specificity and consistency – it ensures that only true antigen-antibody interactions (not “sticky” artifacts) generate the readout signal.

Historically, as soon as ELISA was introduced in the early 1970s (Engvall & Perlmann), the use of Tween‑20 was quickly adopted. Early reports (circa 1970s–80s) showed that washing plates with tap water plus Tween‑20 dramatically improved assay reliability. Today, virtually all ELISA protocols – whether home-brew or commercial kit – utilize Tween‑20 in the wash buffer. Its inclusion is so common that the term “PBST” or “TBST” in a protocol implicitly means PBS or TBS containing Tween‑20. Without Tween‑20, ELISAs often suffer from high background and variability due to proteins sticking to well walls; with Tween‑20 (and proper blocking), the background is kept low and uniform.

Western Blotting

Western blotting involves transferring proteins to a membrane (typically nitrocellulose or PVDF) and probing with antibodies. Tween 20 is indispensable in both the blocking and washing steps. After protein transfer, the membrane is blocked—commonly with 5% non-fat milk or BSA in TBS containing 0.05% Tween 20—to saturate unoccupied binding sites. The detergent helps the blocking protein distribute evenly, prevents drying, and minimizes nonspecific binding “hot spots.”

During antibody incubations, TBST (TBS + 0.05–0.1% Tween 20) is used between steps to wash away loosely bound proteins and reduce antibody sticking to the membrane. This helps retain specific antigen–antibody interactions while removing background noise. As noted in standard references, polysorbate 20 is “a common detergent used in many buffers for washing nitrocellulose membranes.”

If the same membrane needs to be reprobed after initial detection, this Western blot stripping buffer protocol gives a practical walkthrough for removing bound antibodies while preserving the transferred protein signal as much as possible.

Including Tween 20 results in a much cleaner blot: specific bands are sharp and distinct, while nonspecific smears or speckles are reduced. Blotting protocols typically include multiple 5-minute TBST washes after both primary and secondary antibody incubations. Antibody dilution buffers often contain Tween 20 as well, to prevent aggregation and maintain antibody stability.

Tween 20 must be used at optimal concentrations—typically ~0.05%—to effectively reduce nonspecific binding without disrupting true antigen–antibody complexes. Research, especially in autoimmunity, has shown that Tween 20 improves detection specificity in immunoblots. It is also compatible with common detection enzymes such as HRP and alkaline phosphatase, as it doesn’t interfere with their activity at working concentrations.

The incorporation of Tween 20 into Western blot protocols since the 1980s significantly enhanced signal clarity and reproducibility—making it a lasting standard in immunodetection workflows.

Immunohistochemistry (IHC)

Immunohistochemistry on tissue sections relies on antigen–antibody binding within fixed cells/tissues mounted on slides. Tween‑20 is widely used in IHC workflows to promote even staining and reduce background. Typically, IHC protocols employ PBS or TBS with ~0.1% Tween‑20 as a rinse buffer between antibody incubations. A small amount of Tween‑20 (0.01–0.1%) in antibody diluents and rinse buffers assists in uniform wetting of the sample and thorough washing. This is crucial because tissue sections can repel water in spots or dry out; Tween‑20 ensures the reagents spread evenly over the tissue and penetrate all crevices.

Including Tween‑20 in IHC wash buffers also prevents nonspecific sticking of antibodies to tissue components, much like in Western blots. For example, an IHC wash buffer might be PBS + 0.1% Tween‑20 (often called PBST) and multiple washes are done to remove unbound antibody. The result is lower background staining and crisper contrast between specific staining and the surrounding tissue. Tween‑20 has been used as a blocking agent for membrane-based assays at ~0.05%, and similarly, in IHC it can be present in the blocking or serum buffer to saturate nonspecific binding sites on tissue.

Another benefit in IHC is that Tween 20 can enhance reagent penetration in thicker tissue sections. While it is not a strong permeabilizer (see IF section below), its surfactant action helps antibodies and detection reagents to diffuse more uniformly through the tissue surface. Manufacturers of automated IHC stainers often add Tween 20 to their buffers to improve “reagent spreading” on slides. For instance, Biocare Medical’s instructions for an IHC buffer mention that Tween 20’s purpose is to “decrease background staining and enhance reagent spreading in automated and manual procedures”. In summary, Tween 20 is a simple addition that greatly improves the reliability of IHC staining by reducing unwanted background and ensuring even, reproducible staining across the tissue section.

Immunofluorescence (IF) and Cell-Based Assays

Immunofluorescence (IF) and immunocytochemistry on cells share many similarities with IHC in terms of Tween‑20 usage. In IF staining (whether on tissue sections or cultured cells), Tween‑20 is often used as a gentle permeabilizing and washing agent. Unlike IHC, IF typically requires cell membranes to be permeabilized (especially to stain intracellular targets). Common permeabilization agents are Triton X-100 or Tween‑20, with Tween‑20 being the milder option. Tween‑20 at ~0.1–0.2% can permeabilize cellular membranes to some extent while preserving morphology and protein epitopes better than harsher detergents. Triton X-100 tends to fully solubilize membrane lipids (risking extraction of proteins and cell shrinkage), whereas Tween‑20 creates temporary “pore-like” defects in the membrane and has a more renaturing effect on proteins. This gentleness can actually improve antibody access and binding by keeping protein epitopes in a more native state.

In practice, IF protocols might use 0.1–0.5% Tween‑20 in PBS for permeabilization/washing steps when a mild approach is sufficient (for example, if only slight membrane perforation is needed or the target is easily accessible). For instance, an ICC protocol for adherent cells may recommend postfixation washes in PBS + 0.1% Tween‑20 and even use Tween‑20 as the sole permeabilizer if a gentle treatment is desired. Additionally, after permeabilization (whether by Tween‑20 or another detergent), subsequent antibody incubation buffers often include 0.05–0.1% Tween‑20 to keep cells wet and reduce nonspecific staining. Including a bit of Tween‑20 in mounting or washing steps also helps to avoid surface tension effects that can cause uneven staining or drying artifacts on slides.

One caveat: Tween‑20, while milder than Triton, still can affect cells at higher concentrations or longer exposures. Studies have shown that even 0.03% Tween‑20, if left on cells for extended periods (24 h), can induce temporary morphological changes and reduce cell viability – though these effects were reversible upon washing out the Tween. For example, in a virology experiment, treating kidney cells with 0.03% Tween‑20 for 24 hours caused cell membranes to swell and microvilli to decrease, along with a rise in apoptosis markers, but normal morphology returned after the Tween was removed. This indicates that in live-cell contexts, Tween‑20 must be used sparingly. In fixed-cell IF assays, however, these viability concerns are moot, and the focus is on balancing permeabilization with epitope preservation. Researchers often choose Tween‑20 when a “less harsh” permeabilization is needed or in combination with other detergents (e.g., a small amount of Tween‑20 plus saponin).

Beyond immunostaining, cell culture applications for Tween‑20 include using it as a solubilizing agent or to prevent cell clumping. Tween‑20 is generally considered biocompatible at low concentrations and is even tested for cell culture suitability (Sigma’s cell-culture grade Tween‑20 is tested at 0.15 mg/mL ≈ 0.015% for lack of cytotoxicity). In some cases, a tiny percentage of Tween‑20 is added to culture media to help dissolve a hydrophobic drug or compound being delivered to cells. For example, it has been reported that Tween‑20 can reverse multidrug resistance in tumor cells by increasing drug uptake – low concentrations of Tween‑20 enhance the accumulation of chemotherapy drugs in resistant cancer cells. This is attributed to Tween’s ability to mildly perturb the cell membrane or inhibit efflux pumps, thereby allowing more drug to enter and stay in the cells. Similarly, in microbiology, Tween‑20 (or the related Tween‑80) is added to broth to prevent microbial cells from clumping. A small amount (≤0.1%) of Tween can reduce the surface tension and disperse hydrophobic microbial spores or mycobacterial cells that would otherwise aggregate. For instance, preparing spore suspensions of fungi or Bacillus often involves Tween‑20 in water to achieve a uniform single-cell suspension.

In summary, within cell-based experiments, Tween‑20 serves as a gentle detergent that can be used to permeabilize fixed cells for IF, to reduce nonspecific binding in cell staining protocols, and even to aid in delivering compounds to living cells (with caution to avoid toxicity). Its mild nature compared to other detergents makes it valuable when preservation of fine cellular structures or antigen epitopes is important.

Over the decades of Tween‑20’s use, several improvements and optimizations have been made to the reagent itself and how it’s used:

• Stabilization and Shelf-Life: One known issue with polysorbates is their tendency to oxidatively degrade over time, forming peroxides and free fatty acids (especially if exposed to light, heat, or metal ions). Modern solutions to this include adding antioxidants or preservatives to Tween‑20 preparations. Recent research (2020) demonstrated that adding antioxidants like BHT or BHA can protect polysorbate 20 from oxidation, keeping peroxide levels and fatty acid byproducts low during storage. As a result, some commercial Tween‑20 products (especially those used in pharmaceutical formulations) are now formulated with stabilizers to prolong their shelf life and maintain consistency. Additionally, manufacturers recommend storing Tween‑20 at ambient temperature in the dark and avoiding metal contamination to slow any autoxidation. High-purity water and inert containers (plastic rather than glass with metal caps) are used for long-term storage of Tween solutions.
• Sterilization Methods: Early on, some users would autoclave buffer solutions containing Tween‑20 to sterilize them, but it’s now known that autoclaving polysorbate solutions is not recommended – it can cause chemical breakdown and browning of the solution. Over time, protocols have shifted to filter-sterilizing Tween‑20 solutions instead. Suppliers often provide sterile-filtered Tween‑20, and labs will pass Tween-containing buffers through a 0.22 µm filter if sterility is needed, rather than autoclaving. This improvement prevents the loss of detergent activity and formation of harmful byproducts that autoclaving could induce.
• Modified Use in Automation: With the advent of automated staining instruments for IHC/IF and plate washers for ELISA, the use of Tween‑20 has been calibrated for these systems. For instance, automated IHC stainers might use a lower concentration of Tween‑20 in wash buffers to avoid foam formation or reagent pooling, and the timing of Tween-containing wash steps is optimized to ensure no detergent residue is left that might affect mounting media or fluorescence. Some vendors even supply concentrated 20% Tween‑20 stock solutions with instructions specific to manual vs. machine use (adding 2.5 mL to 1 L for manual, or a different ratio for machine that dispenses over many slides). These practical adjustments represent refinements in how the reagent is delivered in modern labs.
• Chemical Modifications: While Tween‑20 itself is a defined chemical mixture, analogous surfactants have been developed. For example, polysorbate 80 (Tween‑80) is used similarly but has a oleate (C18:1) fatty acid and slightly different properties – over time, certain protocols switched between Tween‑20 and Tween‑80 for optimal results (Tween‑20 is more hydrophilic with HLB 16.7, whereas Tween‑80 is a bit more lipophilic, HLB ~15). Improvements also include creation of Tween‑20 alternatives that avoid certain issues (e.g., non-ethylene oxide-based detergents to bypass the formation of PEG-related peroxides). However, no exact “replacement” has superseded Tween‑20; instead, users mitigate its drawbacks by the means above or by switching to a different class of detergents when necessary (like using Triton X-100 if stronger action is needed, or using block copolymer surfactants in some pharma cases).

In essence, the tweaks and improvements have focused on enhancing Tween‑20’s stability and purity, and adapting its use for new technology. These efforts allow Tween‑20 to continue being a reliable component of experiments without introducing artifacts. For example, adding a preservative (like ProClin) to Tween‑20-containing wash buffers in IHC helps prevent microbial growth in the buffer reservoir over time– an issue recognized and fixed in modern labs. Thanks to such refinements, a bottle of Tween‑20 today is more consistent and long-lasting than those in the past, and protocols are optimized to exploit its benefits fully while avoiding pitfalls (like avoiding excessive concentrations or heat exposure).

Advantages of Tween 20:

• Non-Ionic & Mild: Tween 20’s non-ionic nature means it does not disrupt protein structure or protein–protein interactions as harshly as ionic detergents (like SDS). It preserves antigenicity in assays (antibodies retain their binding) and generally does not denature enzymes or ligands at working concentrations. This makes it ideal for assays where maintaining biological activity is crucial.

• Reduces Nonspecific Binding: Perhaps the greatest advantage is its ability to block nonspecific adsorption of proteins to surfaces. By coating well walls, membrane pores, and other surfaces, Tween 20 prevents “sticky” background and improves assay specificity. This leads to clearer results in ELISAs, blots, and histology, as discussed earlier.

• Broad Compatibility: Tween 20 works in a variety of buffer systems (PBS, TBS, etc.) and across a pH range typically used in biology (near neutral pH). It’s compatible with common blocking reagents (serum, BSA, milk), enzymes (HRP, AP), and does not chelate metal ions. It can be used in conjunction with other detergents (e.g., NP-40, Triton X-100) to fine-tune lysis strength. Also, it mixes well with alcohols for special procedures and is soluble in both water and many organic.

• Low Toxicity: Tween 20 is regarded as relatively non-toxic to cells and organisms at the concentrations used. It is even approved as a food additive (emulsifier E432) and used in pharmaceuticals (as an excipient in vaccine and drug formulations). In cell culture, low doses of Tween 20 (≪0.1%) usually do not harm mammalian cells significantly – indeed, some company certifies certain lots for cell culture use. This is an advantage when residual detergent might come into contact with live cells or when considering biocompatibility.

• Cost-Effective and Accessible: Tween 20 is inexpensive and widely available from multiple suppliers. A small volume goes a long way (a liter of 0.05% PBST contains only 0.5 mL of Tween 20). Its long history means it’s referenced in countless protocols, and most labs keep a bottle on hand. The longevity of those bottles is also good; if kept properly, Tween 20 stock can remain effective for years.

• Enhances Reagent Spreading: By reducing surface tension, Tween 20 ensures even coverage of reagents over surfaces and samples. This is an often underappreciated benefit – it prevents “ring effects” when liquids dry and helps washes rinse uniformly. In automated staining, this property helps avoid uneven staining or drying artifacts.

• Versatility: Outside classical immunoassays, Tween 20 finds use in diverse lab techniques – e.g. as a solubilizing agent for membrane proteins, in enzyme assays to keep substrates in solution, in PCR to reduce enzyme sticking, and even in nanoparticle synthesis as a stabilizer. This versatility means one detergent can serve many functions across protocols.

Limitations of Tween 20:

• • Not a Strong Permeabilizer or Solubilizer: Tween‑20’s mildness is a double-edged sword – it is insufficient for lysing cells or solubilizing strong hydrophobic proteins on its own. For instance, it cannot break through nuclear membranes or dissolve integral membrane proteins as effectively as Triton X-100 or SDS. In Western blot stripping buffers or in nucleic acid extraction, stronger detergents are preferred. Thus, Tween‑20 is not suitable when harsh lysis is required; it often needs to be combined with other agents if cell disruption is a goal.
• • Chemical Instability: As noted, polysorbates can undergo oxidation/hydrolysis over time, forming peroxides and acids. Degraded Tween‑20 can become less effective and, worse, the peroxides can oxidize sensitive components (for example, they can inactivate HRP enzyme or oxidize antibodies slightly). Degradation products (fatty acids) might also cause precipitates or haze in protein solutions over long storage. Users must be mindful of storage conditions and avoid using very old or improperly stored Tween that could sabotage an experiment with invisible contaminants.
• • Interaction with Preservatives and Metals: Tween‑20 can bind or sequester certain compounds. It has been reported to reduce the activity of some preservatives in solution. For example, phenolic preservatives or mercurials might partition into Tween micelles, losing efficacy. Heavy metal salts can also cause issues – polysorbates are noted to be incompatible with heavy metal ions and strong oxidizers. If buffers contain metal catalysts (e.g. copper), Tween‑20 can oxidize faster. These interactions limit Tween‑20’s use in some specialized formulations unless countermeasures (like adding excess preservative or chelators) are taken.
• • Biodegradability and Environmental Impact: Tween‑20 is considered relatively biodegradable (especially compared to petrochemical detergents like Triton X-100 which is being phased out due to environmental concerns). However, its polyethylene glycol chains mean it’s not as readily broken down as a simple fatty acid ester. Large volumes of Tween‑20 waste could potentially affect water systems by foaming or altering microbial activity. In lab effluents this is typically negligible, but from an industrial perspective it’s considered moderately biodegradable rather than fully green. This is more of an environmental limitation than a lab performance issue, but it’s driving interest in even more eco-friendly surfactants for the future.