- Western blotting is a widely used method for assessing the specificity of an antibody. The process begins with the preparation of cell lysate, followed by the resolution of proteins on SDS-PAGE, where proteins are separated based on their size. After electrophoresis, the proteins are transferred to a nitrocellulose membrane and subjected to a detection procedure. This involves blocking the membrane with a blocking buffer, followed by treatment with the antibody under investigation, and then secondary antibody. The signal is detected using an appropriate method.
- A clear band at the expected size indicates that the antibody is specific to the protein for which it was generated. However, if the blot shows additional bands, this may suggest that the antibody is recognizing other proteins as well. If the intensity of these additional bands is weaker, it may indicate low affinity, and more stringent conditions could improve specificity. Conversely, if the intensity of the additional bands is similar to that of the specific band, the antibody may not be very specific. Antibodies generated against conserved protein domains are often more likely to recognize multiple proteins.
- Controls are crucial in these experiments. For example, using a knockout cell line for the target protein can be very informative, as no band corresponding to the specific protein should be detected on the Western blot. If a knockout cell line is not available, downregulating the protein using RNA interference (RNAi) can also be effective. In this case, a decrease in the intensity of the specific band in the RNAi-treated cell lysate would be observed.
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