A prion-like protein, Sup35NM-His6, which is safe, easy to produce and able to aggregate into stable amyloid, was developed. This study was to test the feasibility of using Sup35NM-His6 amyloid as a marker for standard sterilization methods known to be effective in inactivating infectious prions. Also Sup35NM-His6 aggregates were spiked into cow brain tissue homogenates to mimic prions in tissues to determine if the aggregate protein could be specifically detected by Western blot at the nanogram level. Like mammalian prions, the proteinase K (PK) resistant fractions of Sup35NM-His6 remain intact after autoclaving. Treatments with 0.1N NaOH or 1.0% NaOCl also resulted in PK undigestible residues. Exposure to the strong denaturant guanidine thiocyanate (> 3 M) destabilized and made the protein PK-digestible. Under strong alkaline conditions of 1.0N NaOH, 2.5 % NaOCl, or a combination of NaOH (0.1 and 1.0N) with autoclaving, Sup35NM-His6 was completely hydrolyzed such that it was no longer detectable by Western blot. Overall, these tests suggest that Sup35NM-His6 could be a useful tool to assess the effectiveness of prion degradation for the prevention of TSE. (c) 2006 Elsevier Inc. All rights reserved.