IHC Primary Antibody Selection & Optimization

Selection and Optimization | Polyclonal Antibodies and Immunohistochemistry | Related Links & Protocols

Primary Antibody Selection & Optimization

Selecting an antibody for Immunohistochemistry staining is important and can affect the experiment’s outcome. Careful consideration of the antibody and its recognized epitope should be taken to ensure the antibody will answer the posed question. Understanding the target protein, its function, tissue and subcellular localization, as well as if it’s subject to post-translational modifications will help determine the choice of antibody. For example, if the protein of interest regulates a protein-protein interaction through its C-terminal end, then an antibody specific for the C-terminus region may be best.
In addition to antibody specificity, the clonality of the antibody should also be considered: monoclonal vs. polyclonal. Both polyclonal and monoclonal antibodies have unique advantages and disadvantages. Understanding their pros and cons in the context of an Immunohistochemistry experiment will inform the choice of antibody.

Polyclonal Antibodies vs. Monoclonal Antibodies:

Polyclonal Antibodies and Immunohistochemistry

Should I use a polyclonal antibody for my IHC experiment? Alteration of protein conformation and tertiary structure can occur as a result of tissue fixation and processing. A change in protein from its native state can impact antibody-epitope binding and affect immunohistochemistry staining. Because polyclonal antibodies are capable of binding multiple epitopes of the same antigen, they are less likely to be affected than monoclonal antibodies by changes in the target protein conformation state.

Is an immunogen affinity purified polyclonal antibody better than whole antiserum? We recommend using an immunogen affinity purified antibody for IHC staining to reduce background staining and increase antibody specificity. Polyclonal antisera is heterogeneous, containing antibodies lacking specificity for the immunizing antigen. Many polyclonal antibodies from Novus are enriched by immunogen affinity purification to enhance specificity for the antigen of interest. Polyclonal antiserum is enriched by passage through an affinity column. Immobilized antigen within the column binds the specific antibody, while non-specific antibody passes through. Elution of the antigen specific polyclonal antibodies results in an enriched antigen-specific population of antibodies.

Optimization of Primary Antibody Incubation

Optimizing your antibody to increase specific staining and decrease non-specific background will improve your IHC staining data. The best antibody concentration, diluent, and incubation time should be determined to achieve the highest specific signal and lowest non-specific background signal.
To begin optimizing, often antibody concentration is varied while incubation time remains constant. To help determine an optimal starting concentration, Novus Biologicals recommends an application specific dilution range for most antibodies. Note that the recommended dilution of immunogen affinity purified antibodies is typically lower than the recommended dilution for monoclonal antibodies (1.7-15 ug/mL polyclonal vs. 5-25 ug/mL monoclonal). This is due to the ability of polyclonal antibodies to bind multiple epitopes of the same antigen. In addition to concentration, incubation temperature and time affect antibody binding and may require optimization. A higher concentration antibody may require shorter incubation time than the same antibody at a lower concentration. It should be noted that longer incubation periods can increase non-specific signal. Therefore, incubation at a lower temperature is recommended If a long incubation period is necessary (i.e. 4 °C versus room temperature). The Novus Biologicals IHC staining protocol recommends an overnight incubation step at 4 °C for most antibodies.