1/23/2024 0 Comments Western blot antibodies![]() What Are the Common Problems with Western Blots? 1. Thanks to their purity and specificity, monoclonal antibodies are known for lower background signals and cross-reactivity than their polyclonal counterparts. The resulting immortalized fused cells, called hybridomas, produce monospecific antibodies that recognize a single epitope on the target antigen. Monoclonal antibodies are generated by fusing an antibody-producing B cell from immunized animals with an immortalized cell line, such as a myeloma cell line. However, because of their heterogeneous nature, polyclonal antibodies tend to give higher background and may cross-react with non-target antigens. Given that one antigen can be bound by multiple antibodies, polyclonal antibodies can provide high levels of sensitivity, which may be advantageous when detecting low abundance proteins. The collected antibodies are often a pool of different immunoglobulin molecules recognizing different epitopes found on the same antigen. Polyclonal antibodies are generated by immunizing laboratory animals (e.g., rabbit) with antigens of interest. The antibodies commonly used in western blotting fall into two main categories: polyclonal and monoclonal antibodies. On the other hand, antibodies that recognize conformational epitopes (e.g., those used in immunocytochemistry and native western blots) may lose binding affinity once target proteins are denatured. Antibodies can recognize epitopes in their denatured linear, primary form (linear epitope), or their native 3D tertiary form (conformational epitope).Īntibodies that recognize linear epitopes under denaturing and reducing conditions (like in SDS-PAGE) may not detect targets whose linear epitopes are concealed in the native protein structure. An antigen normally contains multiple epitopes that can be recognized by different antibodies. An epitope is generally considered to be a stretch of several amino acids. Antibody-Antigen InteractionĪntibody-antigen interactions occur between the antigen-binding site (paratope) of an antibody and a small region on a protein antigen (epitope). Therefore, your antibody choice is critical. Concentration of the target protein is inferred from the intensity of the band that results from the antibody binding to a protein – either through an enzymatic reaction with your antibody or fluorescence from your antibody. Antibodies are then used to detect specific proteins. Following electrophoresis, the proteins are transferred from the gel onto a porous membrane for easy access by blotting antibodies. In the basic western blotting process, polyacrylamide gel electrophoresis (PAGE) separates a mix of proteins according to their molecular weights (denaturing gels) or their 3D structures (native gels). Consequently, high quality antibodies are critical for reliable and consistent western blotting. Successful western blotting means achieving unambiguous results, and this requires a sensitive and specific antibody-antigen interaction.
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