Skip to main content
ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Publications at this Location » Publication #73198
Title: THE USE OF CAPILLARY SDS GEL ELECTROPHORESIS TO DETECT THE PRION PROTEIN EXTRACTED FROM SCRAPIE INFECTED SHEEP.

Author
item Schmerr, Mary Jo
item JENNY, ALLEN - APHIS,NVSL,AMES,IA
item Cutlip, Randall

Submitted to: Capillary Electrophoresis Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/21/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE) that are characterized by neurodegeneration and eventually death. In this study, we examined brains from five sheep that were identified as scrapie positive by immunoblot and immunohistochemistry and five brains from normal sheep that were from a scrapie free flock. We also used this method to follow the extraction and purification of the prion protein. The prion protein was extracted from the brain material by homogenizing the brain stem (10% w/v) in 0.32M sucrose. The particulate material was removed by a low speed centrifugation. The supernatant fluid was subjected to ultracentrifugation at 200,000g. The pellet was resuspended and subjected to treatment with sodium lauroyl sarcosine and proteinase K at a final concentration of 0.01mg/ml. After treatment the solution was centrifuged again at 200,000g. The final pellet was resuspended in 10mM Tris pH 7.4 in a volume of equivalent of 0.1ml/g of brain used. This resuspended pellet was treated with 0.1 M Tris HCl containing 1% SDS and 5% 2-mercaptoethanol and boiled for 10 minutes. The analysis was done in a Beckman P/ACE 5500 and using SDS gel capillary (eCAP SDS 14-200 Beckman capillary). The manufacturer's instructions were followed for preparing the capillary for the size separation. Peaks were observed at 27Kd and at 16Kd from infected sheep but not from normal sheep. This method is a quantitative method to detect and estimate the amount of prion protein that is present in a sample.