Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein

The cellular prion protein (PrP^C) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrP^C proteolysis are of interest because full-length PrP^C and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrP^C may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrP^C C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrP^C proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrP^C using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrP^C and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of β-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrP^C expression levels or proteolysis.