One of the hallmarks of prion diseases is the accumulation of aggregated forms of the prion protein that range in size from few nanometers to particles greater than hundred micrometers. Very little is known about which of these particles are the most pathogenic. Treatments that focus on removing or blocking prion particles are hindered without this information. In this project, we will isolate prion particles from genetic and spontaneous CJD human brain samples using Asymmetric Flow Field-Flow Fractionation (AF4). AF4 is a flow-based separation method with higher resolution range than classical fractionation techniques and particularly useful in the study of protein aggregation diseases. We will use a wide variety of biochemical and biophysical techniques to characterize the isolated human prion particles and determine which of them are the most pathogenic and potential targets for therapeutic intervention in human prion diseases.