Western Blot: Immunodetection - Blocking and Antibody Incubation

Blocking

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. The most commonly used blocking solutions contain 3-5% BSA or non-fat dried milk (also known as Blotto or BLOTTO) in a solution of PBS (phosphate buffered saline) or TBS (tris buffered saline). Often, a small amount of Tween®20 detergent is added to blocking and washing solutions to reduce background staining, and the buffer is known as PBST or TBST. When choosing between these buffers, it is important to note that TBS/TBST is preferred with AP (Alkaline Phosphatase) labeled antibodies because PBS will interfere with the AP signal.

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. However, milk proteins are not compatible with all detection labels, so care must be taken to choose the appropriate blocking solution for the antibodies, buffers, and detection reagents. For example, BSA blocking solutions are preferred with biotin and AP antibody labels, and anti-phosphoprotein antibodies.

This is because milk solutions contain casein, which is itself a phosphoprotein, and biotin, thus it will interfere with the assay results. Commercially supplied blocking solutions, such as Block Ace antibody (BUF029) from Bio-Rad, are very convenient to use and can improve consistency of results, especially when non-specific background signal is an issue.

After blocking, the blot is rinsed in wash buffer, usually TBST, with gentle agitation and in sufficient volume to keep the blot submerged. Please refer to Chapter 5 for detailed protocols.

Antibody Incubation

After blocking and washing, the blot will be incubated in a dilute solution of antibody, usually for a few hours at room temperature or overnight at 4°C. The antibody is diluted in wash buffer (PBST or TBST) or a diluted blocking solution, the choice depends upon the antibody. At Bio-Rad, we offer a HISPEC assay diluent (BUF049A) which can be used with primary and or secondary antibodies to reduce cross-reactivity and minimize non-specific binding. Since antibody preparations vary in their levels of purity and specific binding properties, there will be differences in the level of dilution required.

For example, purified antibodies are usually diluted to a 1-10 mg/ml final concentration. The manufacturer’s datasheet should provide dilution recommendations for a particular preparation. However, it is best to test a range of dilutions with each new antibody, optimizing conditions for the samples under consideration. Dot blots, slot blots, or test blots (see the end of this chapter) can be used for checking various antibody concentrations.

It is critical that all immunodetection steps (blocking, antibody incubation, substrate incubation, and all intervening washes) have a sufficient volume and gentle agitation to keep the blot evenly exposed to the reagents without drying throughout the process.