PBST is phosphate-buffered saline with Tween 20 added, and it is one of the most frequently prepared buffers in any biology lab that runs immunoassays. The “PBS” portion provides a stable, physiological-pH salt solution, while the “T” refers to a small amount of the nonionic detergent Tween 20 (polysorbate 20), typically at a concentration between 0.05% and 0.1%. Its primary job is deceptively simple: wash away proteins and other molecules that are not supposed to be there, without disturbing the ones that are. In practice, getting that balance right involves more subtlety than the recipe suggests.
What Is Actually in PBST
The PBS base is a straightforward aqueous buffer. Most recipes call for sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate dissolved in water, yielding a solution at roughly pH 7.4 with an ionic strength that mimics the inside of a mammalian cell. You can buy it as a pre-mixed powder, as a 10× concentrate, or as a ready-to-use liquid. The buffer keeps the pH from drifting during incubation and wash steps, which matters because antibody-antigen binding is pH-sensitive.
The “T” component, Tween 20, is a polyoxyethylene sorbitan monolaurate. In everyday terms, it is a mild detergent that lowers surface tension and disrupts weak, nonspecific protein-surface interactions. Its nonionic character is the key feature: unlike charged detergents such as SDS, Tween 20 generally does not denature proteins or break apart the three-dimensional structures that antibodies rely on for binding. A typical working concentration of 0.05% (v/v) adds roughly 0.5 mL of Tween 20 per liter of PBS. Some protocols bump that to 0.1%, and a few specialty applications go higher or lower.
Why Tween 20 Is in the Wash Buffer
Every immunoassay depends on specific binding between an antibody and its target. The problem is that proteins are naturally sticky. They adhere to plastic well surfaces, nitrocellulose membranes, and other solid supports through hydrophobic interactions, electrostatic forces, and van der Waals attraction. If those nonspecifically bound proteins include your detection antibody or random serum proteins, your assay produces background noise that obscures the real signal.
Tween 20 tackles this by competing for those hydrophobic binding sites on the solid support. Its molecules wedge into open spots on the plastic or membrane surface, forming a thin layer that prevents other proteins from settling in. At the same time, during wash steps, the detergent helps dislodge proteins that attached weakly. The antibody-antigen interaction you care about is typically strong enough to survive repeated PBST washes, while the nonspecific hangers-on get rinsed away.
Research on ELISA microwells showed that Tween 20 completely saturated untreated polystyrene surfaces at concentrations above about 2 µg/mL, which is well below the 500 µg/mL present in a 0.05% solution. When the wells had been pretreated with poly-L-lysine, however, even high concentrations of Tween 20 failed to fully saturate the surface. In those cases, adding bovine serum albumin (BSA) as an additional blocker was necessary to fill the remaining gaps, and the BSA concentration needed was the same as when BSA was used alone (around 5 µg/mL for complete saturation).1PubMed. Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells That finding matters in practice: if your plate has a specialized coating, you cannot assume PBST washes alone will eliminate background. You may need a dedicated blocking step with BSA, casein, or another protein blocker on top of the Tween 20 in your wash buffer.
PBST in ELISA Workflows
In a sandwich ELISA, a typical protocol involves coating the plate with capture antibody, blocking remaining surface sites, adding the sample, incubating with detection antibody, and then incubating with an enzyme conjugate before adding substrate. Between most of these steps, you wash the wells three to six times with PBST. Each wash floods the well with buffer, and then the liquid is dumped or aspirated. The Tween 20 keeps background low by preventing nonspecific adsorption during each incubation and then stripping loosely bound material during each wash cycle.
Getting the Tween concentration wrong shows up fast. Too little, and the background stays high because nonspecific proteins are not cleared. Too much, and you risk reducing your specific signal because even wanted interactions start to loosen. For most commercial and published ELISA protocols, 0.05% strikes the right balance, and it has become something of a default across the field. Some high-sensitivity assays use 0.1% to push background even lower, accepting a modest drop in signal. If you are optimizing a new assay, titrating the Tween concentration alongside your antibody concentrations is one of the first things to try when background is problematic.
The Tween 20 Stripping Problem in Western Blotting
Western blotting (immunoblotting) uses nitrocellulose or PVDF membranes rather than plastic wells, and this changes how Tween 20 behaves. Early work demonstrated that 0.05% Tween 20 can dissociate antibodies and other proteins directly bound to nitrocellulose. The degree of stripping depends on the specific protein: some antibodies bind nitrocellulose tightly and barely budge, while others are nearly completely removed by the same detergent concentration.2Journal of Immunological Methods. Tween 20 removes antibodies and other proteins from nitrocellulose
This has practical consequences. If your target protein binds the membrane weakly, aggressive washing with PBST can strip it off and destroy your signal. That is why many Western blot protocols use lower Tween 20 concentrations (some drop to 0.02% or even 0.01%) or reduce the number and duration of wash steps compared to ELISA protocols. PVDF membranes have higher protein-binding capacity and tend to hold proteins more tenaciously than nitrocellulose, which is one reason PVDF has become the membrane of choice for many labs. If you are getting weak or disappearing bands despite strong antibody performance in other assays, the wash buffer is one of the first variables to revisit.
Another common issue in Western blotting is using PBST for both blocking and washing. Tween 20 can compete with blocking proteins for membrane binding sites, so adding Tween 20 to a milk-based or BSA-based blocking solution sometimes reduces blocking efficiency. Some protocols deliberately omit Tween from the blocking step and reserve it only for the wash steps, while others include it throughout. The “right” approach depends on your membrane, your target, and your antibody, which is why optimization rather than a single universal recipe remains the norm.
Sodium Azide and the HRP Trap
Many labs add sodium azide (NaN₃) to PBS or PBST as a preservative to prevent microbial growth, especially for buffers that will sit on a bench or in a fridge for weeks. Azide is cheap, effective, and does not usually interfere with antibody function. The problem arises when your detection system uses horseradish peroxidase (HRP), which is one of the most popular enzyme labels in both ELISA and Western blotting.
Sodium azide inactivates HRP through a mechanism-based process. During catalytic turnover in the presence of hydrogen peroxide, azide generates an azidyl radical that irreversibly modifies the enzyme’s heme group. Research showed that roughly 80% of HRP activity is lost after exposure to about 60 equivalents each of azide and peroxide, and higher concentrations completely knock the enzyme out.3PubMed. Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX Even trace amounts of azide carried over in wash buffer can degrade HRP activity enough to weaken or eliminate your signal.
The practical rule is simple: if your assay uses an HRP-conjugated antibody or an HRP-streptavidin conjugate, never use azide-containing PBST for any step that contacts that conjugate. This includes not just the conjugate incubation buffer but also the wash steps immediately before and after. Some researchers use azide-containing PBST for earlier steps (blocking, sample incubation) and then switch to azide-free PBST for the final washes and enzyme incubation. Alkaline phosphatase-based detection systems do not have this vulnerability, so azide is less of a concern if that is your enzyme label.
Shelf Life and Degradation of Tween 20
PBST is often treated as infinitely stable. PBS itself is quite robust: stored at room temperature or in the fridge, it holds its pH and salt concentration for months. Tween 20, though, is a polysorbate, and polysorbates degrade over time through both hydrolysis and oxidation. A review of polysorbate stability in pharmaceutical contexts found that oxidative degradation can be triggered by exposure to light, residual metal traces, peroxides, or elevated temperature, and some of these triggers can be present in the raw material itself.4PubMed Central. Oxidation of polysorbates – An underestimated degradation pathway?
In a research lab rather than a pharmaceutical manufacturing environment, the practical concern is simpler. If you make up a large batch of PBST and leave it on the bench for weeks, the Tween 20 can break down, generating peroxides and free fatty acids. Peroxides are a particular nuisance because they can oxidize sensitive proteins or interfere with enzyme activity. Old bottles of Tween 20 concentrate are also suspect: a bottle that has been open for a year or two, repeatedly exposed to air, may contain enough peroxides to cause unexpected background or signal loss. Some labs test for peroxides using simple colorimetric strips; others simply buy Tween 20 in small bottles and replace it regularly.
For best results, make PBST fresh or store it at 4°C and use it within one to two weeks. If you notice a gradual increase in assay background or a decrease in signal over time and nothing else has changed, old Tween 20 is a surprisingly common culprit. Filtering the buffer through a 0.2-µm filter before storage reduces microbial contamination but does not prevent chemical degradation of the surfactant.
When PBST Interferes with Downstream Work
PBST is designed for immunoassays, and it works well in that context. Trouble starts when residual Tween 20 carries over into other analytical steps. One well-documented example is protein quantification using the Bradford assay. A study examining interfering compounds found that detergents were the most problematic class of interfering substances, causing significant artifacts in Bradford assay readings. The minimum concentration at which interference became measurable varied widely across different detergents and compounds.5ChemRxiv. Shifting focus in the Bradford assay: interfering compounds re-examined If you are washing a sample with PBST and then running a Bradford assay on the eluate, even small amounts of carryover Tween can throw off your protein concentration measurement. A BCA assay is generally more tolerant of detergents, though it has its own sensitivities.
Mass spectrometry is another area where residual Tween creates headaches. High detergent concentrations can suppress ionization and destabilize electrospray, which degrades both sensitivity and data quality.6PubMed Central. Emergence of mass spectrometry detergents for membrane proteomics Detergents also interfere with protease digestion, which is a required step for bottom-up proteomics workflows. If your experiment involves immunoprecipitation with PBST washes followed by mass spectrometry analysis, you typically need extra cleanup steps to remove the Tween 20 before loading your sample. Some labs switch to MS-compatible detergents for the final wash or use detergent-removal spin columns.
The general lesson is that PBST is great at doing one thing: keeping immunoassay surfaces clean. It was not designed to be invisible in every downstream context. Whenever you are carrying a sample from an immunoassay step into a different analytical platform, think about whether residual Tween 20 could be a problem, and plan your cleanup accordingly.
PBS vs. TBS as the Base Buffer
You will sometimes see TBST instead of PBST in protocols, particularly for Western blotting. TBS is Tris-buffered saline, which uses Tris(hydroxymethyl)aminomethane as the buffering agent instead of phosphate. Both buffers maintain physiological pH and similar ionic strength, but the choice between them is not always arbitrary.
Phosphate ions can interfere with alkaline phosphatase-based detection because the enzyme’s substrate is itself a phosphate compound, and excess free phosphate in the buffer acts as a competitive inhibitor. If your Western blot or ELISA uses an alkaline phosphatase conjugate, TBST is the safer choice. For HRP-based detection, either PBST or TBST works fine, and PBS is often preferred simply because it is the more common lab staple.
Phosphate-containing buffers can also cause problems in applications involving divalent metal cations like calcium or magnesium, since phosphate can form insoluble precipitates with these ions. If your protocol requires calcium-dependent binding (some lectin-based assays, for instance), TBS is better. For the vast majority of standard ELISAs and Western blots with HRP detection, PBST and TBST are functionally interchangeable, and the choice comes down to what is already on the shelf and what detection enzyme you are using.
Choosing and Adjusting Your Tween 20 Concentration
The 0.05% default works for most applications, but there are legitimate reasons to go higher or lower. In ELISAs with high background, raising the Tween concentration to 0.1% often helps, since the extra detergent more aggressively competes for surface binding sites. Some cell-based assays and immunohistochemistry protocols also use higher concentrations to permeabilize cell membranes, allowing antibodies to reach intracellular targets. At that point, the Tween 20 is doing double duty as both a wash agent and a mild permeabilization agent.
On the other end, dropping below 0.05% or omitting Tween entirely may be necessary when working with weakly binding antibodies or fragile protein complexes. Some co-immunoprecipitation protocols, for example, use plain PBS for washes because even mild detergent would disrupt the protein-protein interactions being studied. Others use very low concentrations like 0.01% as a compromise. If you are losing your target during wash steps, reducing Tween concentration is a straightforward first troubleshooting move.
A less intuitive consideration is the number and duration of washes rather than the concentration alone. Three quick washes with 0.05% Tween may produce a different result than one long soak at the same concentration. Soaking gives the detergent more time to compete for binding sites, which is helpful for reducing background but also gives it more time to strip weakly bound proteins. For this reason, many optimized protocols specify both the Tween concentration and the wash timing, and changing one without considering the other can shift your results.
Detergent Carryover into Cell-Based Assays
If you are using PBST to wash cells rather than a plastic surface or membrane, the detergent concentration matters for cell viability. At 0.05%, Tween 20 is below the threshold that kills most mammalian cells outright, but prolonged exposure can compromise membrane integrity and affect downstream functional assays. Flow cytometry staining protocols, for example, often include a PBST wash for surface marker staining but switch to plain PBS for intracellular steps where membrane integrity needs to be controlled.
Residual Tween 20 on cells headed into culture is another concern. Even sublethal concentrations of detergent can alter cell signaling, membrane fluidity, and receptor-ligand interactions. Most cell culture protocols that include an immunostaining step are designed so that the staining and washing happen at an endpoint: the cells are being analyzed, not returned to culture. When a protocol does require returning cells to culture after a wash step (certain cell-sorting applications, for instance), plain PBS or a serum-containing medium is preferred over PBST to avoid low-level detergent toxicity.
For fixed and permeabilized cells, as in immunofluorescence microscopy, PBST is a standard wash buffer and the detergent’s effect on living membranes is irrelevant. Here, the Tween 20 helps reduce nonspecific antibody binding to the fixed cellular material and to the glass or plastic surface the cells are mounted on, functioning much as it does in a Western blot or ELISA context.