Blocking/Neutralization Methods, Techniques, Protocols



  • Definition: methods to check antibody specificity or reduce unspecific background staining; used for negative control, competing control, absorption control, isotype control, etc.

  • Demonstration of antibody specificity by antigen (peptide/protein) blocking - It is not uncommon to see more than 1 band in western when probed with a given antibody or see more diffuse staining in immunolocalization studies. The question arises which one of this band(s)/staining is specific. The antibody specificity is generally studied by competing with excess of antigen (peptide or protein) or immuno-neutralization with the antigens.

  • A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue Sections - Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs.

  • Blocking of Unwanted Non-specific Staining for Immunohistochemistry - Source of unwanted staining, besides poor knowledge of the antibody reactivity and malice, is due to: Endogenous enzymes or fluorochromes, Endogenous biotin, Endogenous antibody binding activity (Fc receptors), Crossreactivity of the secondary reagents with endogenous proteins.

  • Specimen Preparation - Blocking - When using antibodies to stain specimens, it is often necessary to block the sample to minimize non-specific binding. Non-specific binding may occur for several reasons: un reacted aldehydes within the preparation may crosslink antibodies to inappropriate structures (especially with glutaraldehyde); highly charged or very hydrophobic structures within the samples may 'trap' antibodies; or, if using polyclonal antibodies, low affinity IgGs may bind speciously to structures that you are not interested in.