Related Links IHC Blocking Non-Specific Binding 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. Polyclonal Antibodies vs. Monoclonal Antibodies:
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Polyclonal Antibodies and Immunohistochemistry |
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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.
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