Comparison of trichloroacetic acid with other protein-precipitating agents in enriching abnormal prion protein for Western blot analysis.
Detection of the abnormal or the pathogenic form of prion protein (PrP(Sc)) by Western blot (WB) is challenging, especially, for samples derived from cell cultures that contain low levels of PrP(Sc). A variety of PrP(Sc) concentration methods have been reported with various PrP(Sc) recovery efficiencies. Ultracentrifugation is one of the methods used frequently to enrich the pathogenic form of PrP(Sc) prior to WB analyses. The resulting PrP(Sc) pellet is extremely insoluble and often requires sonication to be dissolved, potentially generating aerosols. We modified the common protein-precipitating protocol using trichloroacetic acid to concentrate PrP(Sc) by slow-speed centrifugation, followed by solubilization of the pellets with 6 mol/L urea prior to sodium dodecyl sulphate -- polyacrylamide gel electrophoresis and WB analyses. Comparative studies suggest this simple trichloroacetic acid protocol was more effective in enriching PrP(Sc) presented in cell cultures and brain homogenates than other reported protein-precipitating methods. Furthermore, incorporation of the urea treatment step to dissolve the precipitated PrP(Sc) pellets helped to reduce the infectivity of PrP(Sc).
Center for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, ON, Canada.
Matthew
M
LeBrun
Hongsheng
H
Huang
Runtao
R
He
Stephanie
S
Booth
Aru
A
Balachandran
Xuguang
X
Li
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D001921:Brain / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification; D002478:Cells, Cultured; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D011328:Prions / Q000737:chemistry* / Q000302:isolation & purification*; D011489:Protein Denaturation; D012756:Sheep; D014238:Trichloroacetic Acid / Q000032:analysis*; D014508:Urea / Q000032:analysis
D002457:Cell Extracts; D011328:Prions; D014238:Trichloroacetic Acid; D014508:Urea