A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background interference and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.
Ideal blocking agents have the following characteristics:
- Effectively block nonspecific binding of assay reactants to the surface of the well
- Do not disrupt the binding of assay components that have been adsorbed to the well
- Act as a stabilizer (prevent denaturation) of assay reactants on the solid phase
- Do not cross-react with other assay reactants
- Do not possess enzymatic activity that might contribute to signal generation of the substrate or degradation of the reactants
- Perform consistently across various lots
Some of the most commonly used protein blockers are: bovine serum albumin, non-fat dry milk or casein, whole normal serum, and fish gelatin.
BSA: Bovine serum albumin (BSA) is typically used at a 1 to 5% concentration
- An advantage associated with using BSA is its compatibility with protein, BSA is inexpensive and can be stored dry or as a sterile solution at 4°C.
- Disadvantage is cross-reactions with antibodies prepared against BSA-hapten conjugates, lack of diversity required to block some covalent surfaces.
Non-Fat Dry Milk (NFDM) is typically used at 0.1 to 3% concentrations
- NFDM, either homemade or commercial, has a tendency to deteriorate rapidly if not properly prepared and stored. Although casein, a non-fat dry milk component, can be used as a stable blocking reagent (primarily for DNA blots), NFDM tends to be more dispersible in aqueous buffers than pure casein.
- Overall, these are minor issues. NFDM is an excellent blocking reagent. Due to its molecular diversity and amphipathic characteristics, NFDM is the preferred blocking reagent for many covalent surfaces.
- Typically, gelatin is not an adequate blocker when used alone and is actually the least effective biomolecule surface blocker discussed in this bulletin. It blocks mainly protein-protein interactions, sometimes masking specific surface bound proteins and interfering with immunoreactivity. The inferior surface blocking ability and the protein-masking characteristic of gelatin results in higher background and decreased signal.
- The greatest advantage associated with fish gelatin is its lack of cross reactivity with mammalian antibodies and Protein A.
- For extremely difficult blocking problems, the use of normal whole sera at a 10% concentration is recommended.
- The disadvantages of using normal whole sera as a blocking reagent center around its cross-reactivity with Protein A and anti-IgG antibodies.
- Due to its molecular diversity, whole sera effectively blocks: biomolecule-surface (passive adsorption) interactions, biomolecule-covalent surface interactions, and protein-protein interactions, while acting as a protein stabilizer as well.
- Examples of alternative blockers include polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP). These blocking reagents are known for their ability to coat hydrophobic surfaces and render them both non-binding as well as hydrophilic.
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