Caution:  This protocol is subject to the following potential problems: 1.) Example A may require a high concentration of conjugated Fab to achieve effective blocking of the first primary antibody. If this results in unacceptable background, try a lower concentraton of the conjugated Fab followed by further blocking with unconjugated Fab. 2.) If aggregates of conjugated Fab are present and they bind to the first primary antibody, they may act as divalent or polyvalent molecules and capture some of the second primary antibody. This would result in apparent detection of overlapping antigens, even though the antigens may not be overlapping.

Return to Fab Fragments for Blocking and Double Labeling of Primary Antibodies from the Same Host Species

Example B. Use of unconjugated Fab fragments to convert the first, primary antibody into a different species.

Example C. Use of unconjugated Fab fragments for blocking after the first, secondary antibody step.

Example D. Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments.

Example E. Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies.