11/7/2023 0 Comments Flag antibody cross reactivity![]() Inadequate washing may also be a problem when labeling a whole mount or a thick section that requires a longer incubation time. If mouse tissue containing immunoglobulins is probed with a primary antibody made in mouse, the endogenous immunoglobulins should first be blocked with a monovalent Fab fragment of anti-mouse IgG.Īvoid overloading your washing chamber with too many slides. For example, if the tissue is human tonsil and the primary antibody is made in mouse, use a labeled anti-mouse IgG that has been adsorbed against human, such as Alexa Fluor® 488-Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) ( 115-545-062). These principles apply to all immunological procedures including ELISA, Western blots, flow cytometry, immunohistochemistry, and immunocytochemistry.Ĭross-reactivity of labeled secondary antibodies with endogenous immunoglobulins on the tissues or cellsĬhoose a labeled secondary antibody cross-adsorbed against the species of the experimental tissue, if possible. A labeled anti-goat secondary antibody will significantly cross-react with bovine IgG since goat, sheep and cow are closely related species. 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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