Practice question

Co-migration occurs when two proteins of similar molecular weight overlap within same SDS-PAGE band, preventing individual quantification. Developing membrane simultaneously with two primary antibodies leads to superimposed signals unless antibodies are conjugated to spectrally distinct fluorophores. Small interfering RNA knockdown alters physiology and may produce off-target effects. Immunoprecipitation before blotting can separate one antigen but suffers yield loss. Stripping procedure using low pH glycine, SDS, and reducing agent efficiently removes first antibody complex while antigens remain bound, permitting second probing cycle and unambiguous assignment of overlapping bands using same lysate without reloading.