Possible Causes of Weak Signals from Labeled Secondary Antibodies

Signal can be increased by changing from an enzyme- or fluorophore-conjugated antibody to a biotinylated secondary antibody followed by enzyme- or fluorophore-conjugated streptavidin. Some fluorophores are brighter than others, so changing from a FITC-conjugate to Alexa Fluor® 488 will also increase the signal.

A primary antibody that can be detected by a sensitive reporter molecule may not be detectable with a less sensitive reagent. For example, immunoperoxidase is typically 10 to 100 fold more sensitive than immunofluorescence in IHC applications. Using a higher concentration of the primary antibody may solve the problem. Sometimes, an amplification protocol is required to obtain desired signal.

Titrating the primary antibody is recommended for each protocol. The working dilution of a primary antibody may be known for a particular protocol, but may need to be modified if a new detection system is used.

Sodium azide is a potent inhibitor of peroxidase activity. Confirm that azide is not present in buffers or diluents. The water used for reconstitution and the glycerol used to prolong product shelf life may contain peroxidase inhibitors. Assure that dH2O is used for rehydration, and that glycerol is ACS certified.

Jackson ImmunoResearch secondary antibodies are raised against and purified on the solid phase column of immunoglobulins isolated from "normal serum". Therefore, less abundant isotypes may be recognized more weakly. For example, mouse IgG3 is poorly represented in normal serum, and anti-mouse IgG, Fcγ does not recognize it as well as other subclasses. Anti-mouse IgG, subclass specific antibodies provide robust signal from each mouse isotype.

Polyclonal primary antibodies (made in rabbits, goats, guinea pigs etc.) usually contain more than one isotype, so problems with subclass recognition should not be encountered.

Secondary antibodies that have been cross-adsorbed against closely related species (such as anti-mouse adsorbed against rat) recognize only the epitopes on mouse IgG that are different from rat. If a monoclonal mouse IgG primary antibody is not comprised of those unique epitopes, it may be weakly recognized by the labeled secondary antibody.

Carrier proteins such as BSA and dry milk frequently contain IgG. If a labeled anti-goat IgG is diluted in buffer containing IgG-contaminated BSA or dry milk, cross-reactivity of the anti-goat antibody with bovine IgG will remove active antibody from solution and alter the effective concentration. Diluting the antibody in wash buffer only, for example PBST, is preferable to diluting in the presence of carrier proteins. For more effective blocking, incubate with blocking reagent as a separate step instead of adding it to the antibody diluent.