Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane nonspecifically. Blocking is often made with 5% BSA or nonfat dried milk to reduce the background. Nonfat dried milk is often preferred as it is inexpensive and widely available. However, milk proteins are not compatible with all detection labels, so care must be taken to choose the appropriate blocking solution.

• Reduce non-specific binding

Quality Biological's Blocking Solution is prepared from non-fat dried milk in PBS containing sodium azide 0.02% (w/v) as a preservative

It is often a good strategy to incubate the primary antibody with BSA since it is usually needed in higher amounts than the secondary antibody. Putting it in BSA solution allows the antibody to be reused, if the blot does not give good result. BSA blocking solutions are preferred with biotin and AP antibody labels, and antiphosphoprotein antibodies, since milk contains casein, which is itself a phosphoprotein and biotin, thus interfering with the assay results.

Blocking Solution has been used as a reagent to reduce non specific binding of immunological probes in western blotting procedures. Blocking Solution should be used undiluted. Use 0.1 ml of Blocking Solution per cm2 of filter area. A detergent, e.g., Tween 20, may be added to further reduce nonspecific background.