In laboratory settings, achieving accurate results is important, particularly when analyzing biological molecules. Molecules possess an inherent tendency to interact with surfaces or other molecules in unintended ways. These non-specific interactions can compromise the reliability of experimental data. To ensure result integrity, “blocking” is often employed to obtain specific and meaningful measurements.
The Unwanted Sticking Problem
Biological assays often rely on specific molecular recognition, such as an antibody binding to its target protein. However, the surfaces used in these experiments, like plastic plates or membranes, have a natural affinity for proteins and other biomolecules. This inherent “stickiness” means that molecules can attach to these surfaces indiscriminately, rather than exclusively binding to their intended partners. This phenomenon is known as non-specific binding.
Non-specific binding presents a challenge because it leads to background noise, which can obscure the true signal from the specific molecular interactions being studied. High background noise makes it hard to detect the specific binding event. This can result in false positive signals, where a signal is detected even though the specific target is not present, or it can lead to inaccurate quantification of the target molecule. Such false results can compromise the interpretation of experimental data, potentially leading to incorrect conclusions.
How Blocking Buffers Prevent Unwanted Signals
Blocking buffers solve the problem of non-specific binding by saturating all available unoccupied sites on a surface before target molecules are introduced. These buffers contain inert proteins or other compounds that bind to any site where the assay’s detection molecules might non-specifically attach. By coating these surfaces, blocking buffers prevent experimental reagents, such as antibodies, from binding where they shouldn’t.
This process ensures that when specific target molecules are added, their only available binding sites are those provided by their intended partners. For example, if a membrane is coated with a blocking buffer, subsequent antibodies will only bind to the specific protein they are designed to detect, rather than sticking to the membrane itself. Common blocking agents include bovine serum albumin (BSA) and non-fat dry milk, which are protein-based solutions that effectively occupy these non-specific binding sites. The choice of blocking agent can depend on the specific assay and the molecules being studied, as some agents may interfere with certain detection methods or target proteins.
Where Blocking Buffers Are Essential
Blocking buffers are used across numerous laboratory techniques that rely on specific molecular interactions. In Enzyme-Linked Immunosorbent Assays (ELISA), for instance, blocking buffers prevent assay components from binding to the well surface of the plate, which would otherwise lead to high background signals and reduced sensitivity.
In Western blotting, blocking is important to prevent detection antibodies from binding indiscriminately to the membrane after proteins are transferred. This ensures antibodies bind only to specific proteins of interest, resulting in clear bands and accurate detection. Similarly, in immunohistochemistry (IHC) and immunofluorescence (IF), blocking buffers minimize non-specific antibody binding to cellular components or the slide. This reduction in background staining is important for achieving high-quality images and accurately localizing target molecules.