The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be visualized using light microscopy. In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP). It is also used in in situ hybridization (ISH) and sometimes in dot blots and in western blotting. It is most often used in immunohistochemical (IHC) staining as a chromogen. Principles and application of DAB stainingĭAB (3,3′-Diaminobenzidine) is a derivative of benzene. Alternatively, you can assemble the reagents for DAB staining yourself, using biotin-conjugated secondary antibodies, HRP-polymer-conjugated secondary antibodies, and streptavidin-HRP conjugates. Linking HRP and primary antibodies for DAB stainingįor easy results with DAB staining, use a simple DAB substrate kit, or complete DAB staining kit, such as streptavidin-biotin HRP/DAB kits, and polymer method HRP/DAB kits.Principles and application of DAB staining.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. Dot blots, slot blots, or test blots (see the end of this chapter) can be used for checking various antibody concentrations. However, it is best to test a range of dilutions with each new antibody, optimizing conditions for the samples under consideration. The manufacturer’s datasheet should provide dilution recommendations for a particular preparation. Since antibody preparations vary in their levels of purity and specific binding properties, there will be differences in the level of dilution required.įor example, purified antibodies are usually diluted to a 1-10 mg/ml final concentration. 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. The antibody is diluted in wash buffer (PBST or TBST) or a diluted blocking solution, the choice depends upon the antibody. Please refer to Chapter 5 for detailed protocols.Īfter 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☌. 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.Īfter blocking, the blot is rinsed in wash buffer, usually TBST, with gentle agitation and in sufficient volume to keep the blot submerged. This is because milk solutions contain casein, which is itself a phosphoprotein, and biotin, thus it will interfere with the assay results. For example, BSA blocking solutions are preferred with biotin and AP antibody labels, and anti-phosphoprotein antibodies. 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.
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. 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. 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. 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). 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.
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