Optimization of 2D Gel Transblotting of Complex Protein Mixtures

2D gel Western blotting of Host Cell Proteins (HCP) is becoming an important method for characterization of recombinant protein products. However the percentage of protein transferred from a 1D or 2D polyacrylamide gel to PVDF or nitrocellulose membrane depends strongly on conditions of transfer. While optimization of protein transblotting conditions for a few standard proteins is sufficient for many research problems, for HCP samples it is important to maximize recoveries for proteins of varied molecular weights and isoelectric points in complex mixtures. We will present optimization results for 2D gel patterns of protein mixtures based on Coomassie blue staining of PVDF membranes. In addition we will show optimization results based on HCP Western blotting as an assay. Result s for varied buffer pH and transblotting time will be shown.