![]() Post-translational modification or proteolytic processing.Making a fresh sample with sufficient protease inhibitors is advisable. Proteases might digest the target protein during sample preparation or storage. Nevertheless, when the target protein differs in size across species, the target protein may appear at an unexpected molecular weight. Homologous proteins of different species may differ in sizeĪntibodies raised against a protein of one species (e.g., mouse) can often be used to detect the same protein of a different species (e.g.,rat) because the antibodies recognize consensus epitopes present in all homologous proteins.A customized antibody that recognizes a distinct isoform-specific epitope will recognize only the target isoform. If the primary antibody used recognizes an epitope that’s present in the different isoforms, multiple bands may appear on the membrane. Many proteins exist in different isoforms because of alternative splicing. Protein Bands Correspond to Incorrect Molecular Weights The epitope recognized by the primary antibody might not exist in the corresponding protein of different species.Ģ. The primary antibody does not recognize the protein produced in the species of interest.If the primary antibody employed is raised against a native antigen, then it may fail to detect the denatured target protein. Primary antibody cannot bind to epitope on denatured protein samples.The secondary (or detection) antibody should be raised against the species in which the primary antibody is produced (e.g., a rabbit anti-mouse secondary antibody for a primary antibody raised in mice). Incompatibility between primary and secondary 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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