However, most commercially available BSA and dry milk products are contaminated with bovine IgG, which can cause a problem when goat or sheep primary antibodies are used. Two commonly used blocking reagents are bovine serum albumin (BSA) and dry milk. For example, if a primary antibody is made in mouse and normal mouse serum were used for blocking, the mouse IgG would bind to the sticky sites and be recognized by labeled anti-mouse IgG. Never block the tissue or cells with normal serum from the same host species as the primary antibody. The IgG in serum should occupy sticky sites on the tissue or cells to prevent non-specific binding of the labeled IgG antibody. It is most effective to block tissue or cells with normal serum (5% v/v) from the same host species as the labeled secondary or tertiary antibody. Improper blocking of the tissues or cells Not diluting the secondary antibodies far enough.Reactivity of the labeled secondary antibody with immunoglobulins in the diluent.Cross-reactivity of the labeled secondary antibodies with endogenous immunoglobulins on the tissues or cells.Improper blocking of the tissues or cells.Perform a control experiment excluding the primary antibody to isolate the secondary antibody as the cause of background. Background may be caused by the primary antibody.Technical Service: What are common causes of background?
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