Posts tagged ‘2D Gel Electrophoresis’
A few weeks ago we shared with you the first part in a two part series on 2D Gel Electrophoresis. In this last part of the series you will learn: IEF separation, using immobilized pH gradient (IPG) strips, running second-dimension PAGE, spot detection, gel matching, and data analysis, followed by Q&A on topics including sample preparation, storage, and running conditions.
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.
Reliable, Streamlined 2-D Western Blot Workflow for Evaluation of Antibodies Developed for Detection of Host Cell Proteins
The near complete removal of host cell protein (HCP) impurities from biologics (therapeutic protein drugs) is essential for patient safety and treatment efficacy. Hence, biologics are routinely monitored for levels of residual HCPs by a highly sensitive ELISA/immunoassay with a polyclonal antibody reagent raised against a wide range of potential HCP impurities (anti-HCP antibodies). To ensure accuracy of the ELISA, the range of HCP impurities detectable by the anti-HCP antibodies must be evaluated. Although 2-D electrophoresis (2-DE) and western blotting (described in detail in Bio-Rad’s Applications & Technologies section) is the gold standard method for evaluating anti-HCP antibodies, its adoption is hampered by its perceived lack of reproducibility and labor/time-intensive nature of the standard workflow. Here, we demonstrate an improved, optimized workflow for streamlined and highly reliable, 2-DE and western blotting-based evaluation of anti-HCP antibodies.