Blocking Buffer Selection Guide: Tips for Selecting the Best Blocking Agent

Blocking%20agents%20trial%20PBS%20(new)-500x500 Blocking buffers are used to bind the surface areas that may attach to reagents during the testing process and prevent nonspecific antibodies from interacting with reagents. Blockers can also cause poor reading or inaccurate results.

To ensure accuracy in your research, it is important to know how to choose a blocking buffer and learn about the types of available blocking buffers.

What is a Blocking Buffer?

A blocking buffer is a solution of foreign protein, a combination of proteins, or another compound that absorbs all remaining binding surfaces of the plate.

A blocking agent is only useful if it improves the sensitivity of the assay by reducing background interference and improving the signal-to-noise ratio.

Buffers should bind to all potential sites of nonspecific interaction and eliminate the background without altering or obscuring the epitope for antibody binding.

How to Choose a Blocking Buffer

When choosing an appropriate blocking agent, there are three factors you should consider: antibody compatibility, compatibility of the protein of interest, and the detection system.

A buffer should also have the following characteristics:

Effectively blocks nonspecific binding of assay reactants to the surface of the well
Does not disrupt the binding of assay components that absorb to the well
Acts as a stabilizer (prevent denaturation) of assay reactants on the solid phase
Does not cross-react with other assay reactants
Does not possess enzymatic activity that might contribute to signal generation of the substrate or degradation of the reactants
Performs consistently across various lots

Types of Blocking Buffers

There are multiple types of blockers that block specific types of proteins, including animal, fish, and plant-based buffers.

The type of assay and the specific antibodies will determine which type of buffer will produce the best results to prevent false positives from nonspecific antibody interactions.

Plant-Based Blocking Agents

Plant-based blockers, or non-animal based protein blockers, work well for Western blot and ELISA assays. Blockers with non animal based reagents are ideal for immunodetection assays and provide improved sensitivity for best results.

Plant-based blocking agents also offer uniform blocking and reduce staining in the background.

Fish-Based Blocking Agents

Blockers made from fish proteins vary in quality and offer inferior surface blocking ability. Fish-based blockers can interfere with immunoreactivity and mask specific proteins that are bound to the surface.

The G-Biosciences FISH-Blocker utilizes a fish protein as the primary blocking agent. As a non-mammalian protein, fish protein eliminates the interaction of antibodies in mammals. FISH-Blocker is an alternative to milk-based blocking agents and is one of the best blocking agents for immunoassays.

Animal Based Blocking Agents

Animal-based buffers are efficient as long as the proteins they are blocking come from a different animal source. However, you need to find a blocker that binds to all free sites and reduce background noise or clutter.

The sensitivity of the assay also indicates which blocking buffer that will be most efficient. Combined with possible cross-reactivity, you should also consider the surface chemistry if the test is to be successful.

With various types of protein and non-protein animal-based blocking buffers available, it is crucial to choose the correct assay, as it will affect the outcome.

Bovine serum albumin (BSA) is typically used at a 1 to 5% concentration and works well for solid-phase immunoassays. It shows a lack of cross-reactivity and can be stored dry or as a sterile solution at 4°C.

A solution of Nonfat Dry Milk (NFDM) is typically used at a 0.1 to 3% concentration and works best on hard plastic plates. If not adequately prepared or stored, it can deteriorate rapidly. Due to its molecular diversity and amphipathic characteristics, NFDM is the preferred blocking agent for most covalent surfaces.

Non-protein blocking buffers using polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) can perform multiple functions. By hydrophobic coating surfaces, they can be both hydrophilic and non-binding. They can also produce nonspecific backgrounds, even in highly sensitive assays or on diverse surfaces.