Protein blocking agents can be protein mixtures, such as nonfat dry milk or serum, or single proteins such as BSA. Nonfat dry milk is a popular blocking agent because it is inexpensive and easy to find. However, milk contains phosphoproteins that can interfere with blots using anti-phosphoprotein antibodies. Milk can also interfere with biotin-streptavidin based detection systems. In these situations, a blocking buffer containing BSA may be preferred.
Or, to avoid potential cross-reactivity between antibodies and protein blocking agents, a non-protein blocking such as PVP can be used. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. Blocking Buffers for Western Blotting. See Navigation. Before using antibodies to detect proteins that have been dotted or transferred to a membrane, the remaining binding surface must be blocked to prevent the non-specific binding of the antibodies.
Otherwise, the antibodies or other detection reagents will bind to any remaining sites that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target protein or probe components antibodies in the assay can be used for blocking. In practice, however, certain proteins perform better than others because they bind to the membrane or other immobilization surface more consistently or because they stabilize the function of other system components.
Purpose and function of blocking steps Types of blocking buffers Which blocking buffer to use? Due to the number of proteins within milk, milk may mask some antigens and lower the detection limit of the western blot. BSA is generally a weaker blocker, which can result in more non-specific antibody binding, but can increase the detection sensitivity for low-abundant proteins.
Purified proteins e. Ideal when blockers, such as non-fat milk, block antigen-antibody binding More expensive than traditional non-fat milk formulations. Click image to enlarge. Recommended Thermo Scientific western blot blocking buffers. Blocking agent Highlights When to use Available formats Pierce Clear Milk Blocking Buffer Clarified and stabilized milk proteins High-performance replacement for homemade milk blocking buffers Long shelf-life at room temperature Use when high background seen with Non-fat milk Fluorescent and chemiluminescent applications Borate, pH 7.
DenHollander, N, Defus D. J Immunol Methods 1 — Education center homepage. Western blotting protocols. Western blotting webinars. For Research Use Only. Not for use in diagnostic procedures. Create mode — the default mode when you create a requisition and PunchOut to Bio-Rad. You can create and edit multiple shopping carts. Edit mode — allows you to edit or modify an existing requisition prior to submitting. You will be able to modify only the cart that you have PunchedOut to, and won't have access to any other carts.
Inspect mode — when you PunchOut to Bio-Rad from a previously created requisition but without initiating an Edit session, you will be in this mode. You cannot modify any Cart contents. Click here to find out how. Blocking is a very important step in the immunodetection phase of Western blotting because it prevents non-specific binding of antibody to the blotting membrane.
Non-fat dried milk is considered to be a good starting point when selecting a blocking solution because it is inexpensive and in very wide use.
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