Benefits and pitfalls of secondary antibodies: why choosing the right secondary is of primary importance.
Simultaneous labeling of multiple targets in a single sample, or multiplexing, is a powerful approach to directly compare the amount, localization and/or molecular properties of different targets in the same sample. Here we highlight the robust reliability of the simultaneous use of multiple mouse monoclonal antibodies (mAbs) of different immunoglobulin G (IgG) subclasses in a wide variety of multiplexing applications employing anti-mouse IgG subclass-specific secondary antibodies (2°Abs). We also describe the unexpected finding that IgG subclass-specific 2°Abs are superior to general anti-mouse IgG 2 °Abs in every tested application in which mouse mAbs were used. This was due to a detection bias of general anti-mouse IgG-specific 2°Abs against mAbs of the most common mouse IgG subclass, IgG1, and to a lesser extent IgG2b mAbs. Thus, when using any of numerous mouse mAbs available through commercial and non-profit sources, for cleaner and more robust results each mAb should be detected with its respective IgG subclass-specific 2°Ab and not a general anti-mouse IgG-specific 2°Ab.
Department of Neurobiology, Physiology and Behavior, University of California Davis, Davis, California, United States of America.
Colleen F
CF
Manning
Angeliki M
AM
Bundros
James S
JS
Trimmer
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D001921:Brain / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D051381:Rats
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007147:Immunoglobulin Light Chains