The Development of an Efficient Immuno-Slotblotting Technique to Quantify the Effects of Protein Disulfide Isomerase on Prion Protein Misfolding
The objective of this project was to develop an efficient immuno-slotblotting technique that could be used as a quantitative assay in measuring the effects of Protein Disulfide Isomerase (PDI) on prion protein misfolding in Chronic Wasting Disease (CWD). Chronic Wasting Disease is a transmissible spongiform encephalopathy that is horizontally transmitted amongst families of cervids, which includes elk, deer, and moose. The infectious agent in CWD is a misfolded prion protein that comes into contact with other prion proteins, causing them to misfold. Previous observations have suggested that disulfide bond rearrangement may be an important step in the misfolding process. In order to measure rates of prion misfolding, a method must be developed to detect the prion protein in a quantitative way. Previous attempts to quantify the amount of prion protein using an immuno-slotblot technique have encountered problems. The technique developed here uses limiting dilution, immuno-slotblot analysis through the use of a Bio Dot SF Microfiltration apparatus. The immuno-slotblot analysis is found to be more sensitive than conventional immunoblotting and can be run following a methanol precipitation of protein to maximize results. This technique can be used to measure small amounts of prion proteins in large volume samples using an immuno-slotblot. Also, a technique involving Proteinase K digestion was found to be effective at efficiently detecting prion proteins in smaller samples. This technique can be used to measure the effect of PDI on the misfolding of CWD prion proteins.