Best Blocking Buffer Selection for ELISA & Western Blot

Antibodies are used to detect specific proteins during research. There are a variety of tests including the Western blot, that produce a surface area that can contain other proteins and components that need to be blocked. Blocking buffers are used to bind the surface areas that may attach to reagents during the testing process, preventing the non-specific antibodies from interacting with the reagents and causing a poor reading or inaccurate results. Knowing which buffers to use, however, is critical for ensuring the accuracy of a test, as each option has its own advantages and disadvantages.

Types of Blocking Buffers

There are several types of blocking buffers that are used to isolate proteins. Some of the most common of which include animal, fish, and plant-based buffers. Each one of these options blocks specific types of proteins. The type of assay and the exact antibodies that are being targeted will determine which type of buffer will produce the best results to prevent false positives from non-specific antibody interactions. Here’s how to decide between them.

Plant Based Blocking Buffers

Plant or non-animal based protein blockers work well for Western blot and ELISA assays. Blockers with based reagents are excellent for immunodetection assays and provide improved sensitivity for optimum results. These blocking buffers offer uniform blocking across the board and reduce staining in the background.

Fish Based Blocking Buffers

Blocking buffers made from fish proteins tend to vary in quality and may offer inferior surface blocking ability. These blockers can interfere with immunoreactivity and can mask certain proteins that are bound to the surface. The greatest benefit it has to offer is that it offers minimal cross-reactivity with Protein A and antibodies derived from animals. Blockers made with fish proteins have been reported to be extremely inadequate when used alone and can result in decreased sensitivity.

Animal Based Blocking Buffers

Blocking buffers derived from animal sources work well as long as the proteins they are blocking comes from a different animal source. The key is to find a blocker that will bind to all free sites and reduce any background "noise" or clutter. Bovine serum albumin is excellent for solid phase immunoassays, while a solution of non-fat dry milk powder works best when used on hard plastic plates. Certain formulations of non-fat dry milk powder can inhibit alkaline phosphatase activity as well as cause reactions with anti-phosphotyrosine antibodies.

Non-protein blocking agents using polymers like polyvinyl alcohol (PVA), polyethylene glycol (PEG), and polyvinylpyrrolidone (PVP) are reagents that can perform several different functions. By coating hydrophobic surfaces, they can cause them to be both hydrophilic and non-binding. They can also produce backgrounds that are non-specific, even on the most diverse surfaces and in highly sensitive assays.

When a blocker is chosen, several things must be considered including lot-to-lot variations, the surface on which the assay is performed, and the types of antibodies and proteins involved. While the goal is to identify specific antibodies, it is also important to reduce the impact of the background and prevent proteins and other molecules from disrupting the quality of the test. The types of proteins as blocking reagents must be different from those being identified. The ability to block the membrane on the surface must be sufficient enough to reduce signal-to-noise or clutter when it comes to reading the final test. 

The sensitivity of the assay will also indicate which type of blocking buffer will be most efficient. The surface chemistry, combined with possible cross-reactivity must also be considered if the test is to be successful. With the many different blocking buffers available, both protein and non-protein, choosing the right one for a particular assay is extremely important and will have a dramatic effect on the final outcome.