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Benefits and pitfalls of secondary antibodies: why choosing the right secondary is of primary importance.

PloS one | Jun 7, 2012

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.

Pubmed ID: 22675541 RIS Download

Mesh terms: Animals | Antibodies, Monoclonal | Antibody Specificity | Brain | Fluorescent Antibody Technique | Humans | Immunoassay | Immunoblotting | Immunoglobulin Heavy Chains | Immunoglobulin Isotypes | Immunoglobulin Light Chains | Mice | Mice, Knockout | Rats

Research tools detected in this publication

Data used in this publication

None found

Associated grants

  • Agency: NINDS NIH HHS, Id: NS34383
  • Agency: NINDS NIH HHS, Id: R01 NS042225
  • Agency: NINDS NIH HHS, Id: R01 NS034383
  • Agency: NINDS NIH HHS, Id: NS42225
  • Agency: NINDS NIH HHS, Id: U24 NS050606
  • Agency: NINDS NIH HHS, Id: NS50606
  • Agency: NINDS NIH HHS, Id: R37 NS034383

Antibody Registry (Reagent, Antibodies)

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A national mouse monoclonal antibody generating resource for biochemical and immunohistochemical applications in mammalian brain. NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mouse mAbs that are optimized for use in brain (i.e. NeuroMabs): for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks. The NeuroMab facility was initially funded with a five-year U24 cooperative grant from NINDS and NIMH. The initial goal of the facility for this funding period is to generate a library of novel NeuroMabs against neuronal proteins, initially focusing on membrane proteins (receptors/channels/transporters), synaptic proteins, other neuronal signaling molecules, and proteins with established links to disease states. The scope of the facility was expanded with supplements from the NIH Blueprint for Neuroscience Research to include neurodevelopmental targets, the NIH Roadmap for Medical Research to include epigenetics targets, and NIH Office of Rare Diseases Research to include rare disease targets. These NeuroMabs will then be produced on a large scale and made available to the neuroscience research community on an inexpensive basis as tissue culture supernatants or purified immunoglobulin by Antibodies Inc. The UC Davis/NIH NeuroMab Facility makes NeuroMabs available directly to end users and is unable to accommodate sales to distributors for third party distribution. Note, NeuroMab antibodies are now offered through antibodiesinc.


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Common Fund Protein Capture Reagents

Program that is developing new resources and tools to understand the critical role the multitude of cellular proteins play in normal development and health as well as in disease. These resources will support a wide-range of research and clinical applications that will enable the isolation and tracking of proteins of interest and permit their use as diagnostic biomarkers of disease onset and progression. The program is being implemented in phases, with three Funding Opportunity Announcements (FOAs): * FOA 1: Antigen Production (RFA-RM-10-007) To produce human transcription factor antigens for making monoclonal antibodies or other affinity capture reagents; this effort is already underway. * FOA 2: Anti-Transcription Factor Antibodies Production (RFA-RM-10-017) To optimize and scale anti-transcription factor capture reagent production to develop a community antibody resource. * FOA 3: New Reagent Technology Development and Piloting (RFA-RM-10-018) To develop improvements in the reagent production pipeline with regard to quality, utility, cost, and production scalability. To understand what makes a cell function normally and what may go awry in disease, we need better tools and resources, such as renewable protein capture reagents and probes, to study how proteins work in isolation and how they interact with other proteins, carbohydrates, or DNA regions within a cell. Ideally, this resource would allow us to identify and isolate all proteins within cells, in their various forms the so called proteome to ensure broad application in research and clinical studies aimed at understanding, preventing, detecting and treating disease. Existing protein capture reagents, such monoclonal antibodies, have been developed for a number of protein targets, although these represent only a subset of all proteins comprising the human proteome. In addition, many monoclonal antibodies lack the desired level of specificity and do not reliably target only the protein of interest. This is particularly problematic given the multiple forms of any one protein and the broad range of protein types in the body. The Protein Capture Reagents Program is organized as a pilot program using transcription factors as a test case to examine the feasibility and value of generating a community resource of low cost, renewable affinity reagents for all human proteins. The reagents must be specifically designed for high quality and broad experimental utility in order to meet the growing demands of biomedical researchers. Based on what is learned from these funding initiatives, the program may expand to a larger production effort to provide a broad community resource of human protein capture reagents.


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