"Without question, I can always turn to Jackson ImmunoResearch secondary antibodies for ECL detection of any primary antibody! They produce great signal at low dilutions."
W Thompson, OSHU
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Caution: This protocol may be subject to the following 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 concentration of the conjugated Fab, followed by further blocking with unconjugated Fab.
(2) If small aggregates of conjugated Fab are bound to the first primary antibody, they may act as divalent or polyvalent antibodies and capture some of the second primary antibodies, which would result in overlapping detection of antigens.