Thermo Scientific Blocker BLOTTO Blocking Buffer is a ready-to-use 5% solution of nonfat powdered milk in Tris-buffered saline for Western blot, ELISA, IHC and other detection methods.
- Popular – nonfat milk has been used for many years in a variety of protein methods, although it is not recommended for avidin-based techniques because it contains some endogenous biotin
- Convenient – supplied as a ready-to-use 1X TBS solution; can be diluted as needed
- Easy to use – formulated with anti-foaming agent and thimerosal-free preservative
- Flexible – may be used for multiple applications, including as a diluent for antibody
The purpose of the blocking step in an assay is to improve assay sensitivity by reducing background interference. However, unforeseen cross-reaction of detection reagents with blocking buffers is itself a cause of high background and low signal-to-noise ratios in assay systems. Because individual blocking buffers are not compatible with every system, a variety of blockers in both Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) are available. The best blocking buffer for a specific experiment will bind to all potential sites of nonspecific interaction, eliminating background without altering or obscuring the epitope for antibody binding.
To optimize the blocking step for a particular immunoassay, empirical testing is essential. Using inadequate amounts of blocker will result in excessive background. Using an excessive blocker concentration can mask antibody-antigen interactions or inhibit the marker enzyme. For best results when developing a new immunoassay, test several different blocking agents for the highest signal-to-noise ratio in the assay. There is no single blocking agent that is ideal for every occasion because many factors can influence nonspecific binding, including various protein interactions unique to a specific assay system.
- Alegria-Schaffer, A., et al. (2009). Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol. 463:573-99.
- Abrams, E.T., et al. (2003). J. Immunol. 170:2759-64.
- Goretzki, L., et al. (2000). J. Biol. Chem. 275:28625-33.
Blocking buffers for Western blot and ELISA -- technical guide
Blocking strategies for immunohistochemistry -- technical guide
Pierce Clear Milk Blocking Buffer (10X)
All Blocking Buffers