Phosphoprotein Blocking Buffer 1000ml

Phosphoprotein blocking buffer is a blocking buffer designed for western blot for phosphoprotein. For optimal result, the antibodies must bind only to the protein of interest and not to the membrane. Non-specific binding (NSB) of antibodies can be reduced by blocking the unoccupied membrane sites with an inert protein or non-ionic detergent. The blocking agent should have a greater affinity for the membrane than the antibodies. It should fill all unoccupied binding sites on the membrane without displacing the target protein from the membrane. The most common blocking agents used are bovine serum albumin (BSA, 0.2?5.0%), non-fat milk, casein, gelatin, and dilute solutions of Tween?-20 detergent (0.05?0.1%). Tween-20 detergent was also shown to have a renaturing effect on antigens, resulting in improved recognition by specific antibodies (Van Dam et al., 1990; Zampieri et al., 2000). Other detergents, such as Triton? X-100 detergent, SDS, and NP-40, are sometimes used but can be too harsh and disrupt interaction between proteins.The blocking agent is usually dissolved in PBS or TBS buffers. There are risks associated with blocking; a poorly selected blocking agent or excessive blocking can displace or obscure the protein of interest.Therefore, the correct choice of a blocking agent can be critical to a successful immunodetection. For example, dry milk cannot be used with biotinylated or concanavalin-labeled antibodies, since milk contains both glycoproteins and biotin. The analysis of phosphorylated proteins with phospho-specific antibodies can be compromised if using crude protein preparation as a blocking agent. These preparations may contain phosphatases, and the phosphorylated proteins on the blot could become dephosphorylated by this enzyme.