NR AKNM
AU Schmerr,M.J.; Jenny,A.; Cutlip,R.C.
TI Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep
QU Journal of Chromatography. B, Biomedical Science and Applications 1997 Sep 12; 697(1-2): 223-9
PT journal article
AB Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to conventional SDS-PAGE and Western blot to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10%, w/v) in 0.32 M sucrose and by using a series of ultracentrifugation steps and treatment with sodium lauroyl sarcosine and proteinase K. After the final centrifugation step, the pellet was resuspended in 0.01 M Tris pH 7.4 in a volume equivalent to 0.1 ml/g of brain used. This resuspended pellet was treated with 1% SDS and 5% 2-mercaptoethanol and boiled for 10 min. The analysis was done in a Beckman P/ACE 5500 using a SDS gel capillary (eCap SDS14-200 Beckman capillary). In infected sheep brain samples, but not normal sheep, a major peak at a molecular mass of 16.1 kDa and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimated on Western blot (22.4 kDa), a Ferguson plot was made to determine if there were abberations in the molecular mass determination. After correction, the major peak was estimated to be 19.2 kDa. This has a better correlation with that determined by SDS-PAGE and Western blot. The equivalent amount of brain sample in the capillary was approximately 50 micrograms. For Western blot, the amount of brain sample was approximately 20 mg. For this assay, this is approximately 100 times less than that needed for Western blot for sheep samples.
IN Die Autoren verglichen die Sensitivität der SDS-Kapillargelelektrophorese mit der Sensitivität konventioneller Western Blots hinsichtlich des Nachweises proteaseresistenten Prionproteins. Während sie für den Western blot 20 mg Gehirn brauchten, reichten bei der SDS-Kapillargelelektrophorese 50 µg. Damit war die SDS-Kapillargelelektrophorese 400-fach empfindlicher.
ZR 21
MH Animal; Blotting, Western; Brain/metabolism/virology; Comparative Study; Electrophoresis, Capillary; Electrophoresis, Polyacrylamide Gel; Gels; Prions/*analysis; Scrapie/*metabolism/virology; Sheep; Sodium Dodecyl Sulfate
AD National Animal Disease Center, Ames, IA 50010, USA
SP englisch
PO Niederlande