Antibody Applications


Our Immunohistochemistry (IHC) Laboratory screens antibodies for suitability for use in IHC applications, selecting only the antibodies that give the best signal to background ratio. A representative tissue is selected and a standard protocol used to assess both monoclonal and polyclonal antibody suitability. Antigen affinity-purified polyclonal antibodies maximize the opportunity of antigen binding in tissue, due to multiple epitope recognition but without the drawback of conventional polyclonal antibodies (non-specific binding).

Detailed IHC protocols have been developed and optimized by our in-house laboratory, using frozen and paraffin-embedded tissues and either chromogenic or fluorescent detection methods. The protocols are available on our website and can be modified to suit your tissue sample. Paraffin-embedded tissues may require antigen retrieval, depending on the tissue type, fixation protocol, and storage.

Images of IHC staining are available on the website. Each image is accompanied by details of the protocol used, antibody concentration, and antigen retrieval (if used).

Additional Reagents for Immunohistochemistry

Western Blot

The majority of both monoclonal and polyclonal antibodies are validated for use in Western blot. In-house quality control uses Western blot applications to show that antibodies are sensitive and specific for the protein to which they are raised. Proteins are run under reducing and non-reducing conditions.

Antigen affinity-purified polyclonal antibodies (Cat. # AFXXX) offer maximum opportunity to detect your sample through multiple epitope recognition without the drawback of conventional polyclonal antibodies (non-specific binding). Many of these antibodies are also available biotinylated, eliminating the need for a secondary antibody and offering the flexibility to choose your preferred detection system.

A suggested protocol for Western blot is available on our website along with an extensive troubleshooting guide.

Additional Reagents for Western blot

Polyclonal and monoclonal IgG preparations are available for use as negative controls in Western blots.
Molecular weight markers are also available biotinylated or pre-stained blue.

ELISA Development

R&D Systems offers a wide range of matched antibody pairs intended for ELISA development. Each pair has been tested to ensure:

  • Compatibility in forming a sandwich with the analyte
  • Stability once opened and reconstituted
  • No cross-reactivity with related molecules

Detection antibodies are offered biotinylated for flexibility to choose any colorimetric, fluorescent, or chemiluminescent detection system for which streptavidin conjugates are available.

Additional Reagents for ELISA Development


Neutralizing a particular protein with antibodies can give valuable information about a protein’s biological activity, whether it is used in vivo or in vitro. Neutralization is also essential in non-specific bioassays when measuring the biological activity of samples containing several bioactive proteins.

Selected R&D Systems’ antibodies are tested for their ability to neutralize the biological activity of their target protein. To test neutralization, a concentration of protein, just high enough to elicit a maximum biological response, is incubated with increasing concentrations of antibody. The concentration of antibody which causes 50% neutralization of the biological response is termed the ND50. Low ND50 values indicate potent neutralization. Each new lot of antibody is assayed to ensure the ND50 value is consistent between lots. Details of the bioassay used and the neutralization effect are stated on all product datasheets for neutralizing antibodies. Low endotoxin, no preservatives, and high antibody specificity ensure that the observed neutralization of bioactivity is specifically due to antibody binding of target protein.

The ND50 of monoclonal anti-mouse EPO (Catalog # MAB959) was determined to be approximately 0.2–0.8 microgram/mL in the presence of 40 ng/mL of rmEPO using the factor-dependent cell line TF-1 which proliferates in response to rmEPO. 3H-thymidine was added for the final 4 hours of a 72-hour incubation to determine proliferation.

The ND50 of the monoclonal anti-human BLyS (Catalog # MAB124) was determined to be approximately 2–10 microgram/mL when using rhBLyS at 2.5 ng/mL and 4 x 106 primary B cells, which proliferate in response to rhBLyS. 3H-thymidine was added for the final 16 hours of a 72-hour incubation to measure cell proliferation.

Flow Cytometry

Flow cytometry measures the light excitation and scattering characteristics of individual cells to assess certain physical and chemical characteristics of cells. Rapid analysis of large numbers of cells, analysis of several parameters on the same cell, and the collection of quantitative data on a single cell are some of the advantages that flow cytometric analysis offers over other technologies.

R&D Systems offers a wide range of antibodies conjugated to different fluorochromes that allow for simultaneous multiparameter analysis. These directly conjugated antibodies are designed for monitoring the presence of specific proteins or other target structures on either cell surfaces or intracellularly. Cell surface staining is useful in the immunophenotyping of cell populations, while intracellular cytokine staining has been utilized to identify cells secreting selected cytokines, e.g. Th1 vs Th2 cells.

Intracellular staining antibodies are selected from a panel of monoclonals or, in some cases, carefully selected polyclonals for their specific recognition of cytoplasmic forms of molecules. All directly conjugated antibodies can also be used to perform quantitative flow cytometry analysis to estimate the number of molecules expressed on each cell.

Flow cytometry qualified unlabeled primary antibodies are also available with a choice of labeled secondary antibodies. Use our antibody search tool to identify antibodies for specific analytes.

Additional Reagents for Flow Cytometry

A variety of fluorochrome labeled isotype matched controls, conjugated to the same fluorochrome/protein ratio, are also available from R&D Systems as an aid in assessing the background staining of cell populations.