Tris-Buffered Saline with Tween 20, commonly known as TBST, is a fundamental reagent used extensively in molecular biology and diagnostic laboratories. TBST is a specially formulated liquid solution combining buffering agents, salts, and a mild detergent. Its primary purpose is to act as a wash buffer in complex testing procedures. By maintaining stability and removing unwanted materials, TBST ensures that only the specific biological interactions of interest are measured, generating accurate and reliable results for health-related research and diagnostic tests.
The Role of Each Component in the TBST Buffer
TBST is a composite solution, and each of its three main components performs a distinct biological function. Tris, the buffering agent, maintains a stable pH level, typically between 7.4 and 7.6. This stable pH is necessary because proteins, like the antibodies used in testing, can lose their proper structure and function if the environment becomes too acidic or too alkaline. Tris is often preferred because it does not form complexes with common ions like calcium or magnesium, which can interfere with certain assays.
The second component is saline, a solution of sodium chloride (NaCl) that mimics the physiological environment. This salt content maintains isotonicity, balancing the osmotic pressure inside and outside biological components. Maintaining this balance prevents cells from shrinking or swelling, ensuring their structural integrity is preserved during the testing process.
The third component, Tween 20, is a mild, non-ionic detergent that distinguishes TBST from Tris-Buffered Saline (TBS). Tween 20 is typically included at a low concentration to disrupt non-specific attractions between molecules. This detergent promotes a thorough and effective wash, enhancing the purity of the signal detected in the final results.
How TBST Prevents Background Noise in Testing
The primary role of TBST is to minimize “background noise,” which refers to false positive signals in molecular assays. This noise is caused by non-specific binding, where antibodies or detection probes attach weakly to the testing surface instead of their intended target. If these unwanted molecules are not completely removed, they can generate a signal that obscures the true result, leading to an inaccurate reading.
The detergent action of Tween 20 is central to solving this problem by acting as a surfactant. Tween 20 works by reducing surface tension and disrupting the weak hydrophobic interactions that cause molecules to stick to the testing surface. The detergent molecules surround and lift these loosely bound, non-target proteins and excess reagents from the surface without disturbing the strong, specific antigen-antibody complexes that are the focus of the test.
TBST is used repeatedly throughout the testing protocols in a series of washing steps following each incubation phase. This methodical rinsing process flushes away the dislodged, non-specifically bound material. By ensuring that the only molecules remaining on the surface are those that have specifically and strongly bound to their targets, TBST significantly improves the signal-to-noise ratio of the assay. This stringent washing procedure makes it possible to obtain clear, high-contrast results necessary for accurate scientific interpretation.
Key Applications of TBST in Health Research and Diagnostics
TBST’s role as a high-performance wash buffer makes it indispensable across several major techniques used in health research and clinical diagnostics. One of its most common applications is in Western Blotting, a technique used to identify specific proteins in a complex sample, such as detecting a specific disease biomarker. TBST is the standard buffer used to wash the membrane after the primary and secondary antibodies have been applied, ensuring only the target protein bands are visible.
TBST is also a widely used reagent in the Enzyme-Linked Immunosorbent Assay (ELISA), a method that quantifies substances like hormones, antibodies, or antigens in a sample. During an ELISA, TBST is used to wash away all unbound components between steps, preventing background interference that could skew the concentration measurements. The ability of TBST to provide a clean background is important in diagnostic tests that rely on measuring very low concentrations of a target molecule.
The buffer extends its utility into tissue analysis through techniques like Immunohistochemistry (IHC) and Immunofluorescence (IF). These methods are used to visualize specific proteins within tissue sections. TBST is incorporated into the preparation and washing steps to prevent antibodies from sticking non-specifically to the tissue components. This application ensures that the resulting images show precise localization of the target molecule, which is necessary for pathology and cancer research.