Using Bio-Rad’s PDQuest™ Software to Generate a Match Rate between 2-D Electrophoresis and Western Blotting
Two-dimensional (2-D) electrophoresis is a widely used gel-based method for separating protein mixtures by charge (isoelectric point) and size (molecular weight). After 2-D electrophoresis (2-DE; described in detail in the Bio-Rad Applications & Technologies section), gels can be processed for protein detection via total protein staining and/or western blotting. Western blotting analysis following 2-DE separation is particularly useful in at least two important applications: (1) identification of pathogen-derived proteins that are highly immunogenic (have potential as vaccine candidates), and (2) evaluation of the range of host cell protein (HCP) impurities detectable by polyclonal antibodies used to monitor HCP impurities in purified biopharmaceuticals. For both applications, it is critical to generate a match rate from an overlay of the 2-DE and western blotting analyses. Bio-Rad’s PDQuest 2-D analysis software can be used to easily generate match rates. We describe here, step-by-step guidelines for generating this information using PDQuest software.