PMCA substrate homogenates were prepared from mice that had been perfused with PBS containing 5 mM EDTA at the time of death. PrP-null (Prnpo/o), Tg35 (homozygous for huPrP G127/M129), Tg152 (homozygous for huPrP G127/V129) or Tg183 (homozygous for huPrP V127/M129)) mouse brains25 (link) were homogenised in cold conversion buffer (PBS containing 150 mM NaCl, 1.0% (v/v) Triton X‐100, 4 mM EDTA and 1× Complete protease inhibitor (Roche Applied Science)), using a Duall tissue grinder to give a 10% (w/v) homogenate.
Substrates were seeded with a 1/100 dilution of vCJD (I4618) 10% brain homogenate in PBS. Each reaction mixture was divided in two prior to PMCA with one half stored at −70 °C as a minus PMCA control. PMCA consisted of 96 cycles of 30 s sonication every 30 min in a Misonix S3000 at 75% power output (Misonix, Farmingdale, NY), reactions were carried out with 40 µl substrate in 200-µl thin-walled PCR tubes at 35 °C.
Samples were digested with 50 µg ml−1 proteinase K (PK) for 1 h at 37 °C. The reaction was stopped by the addition of AEBSF in SDS-loading buffer and samples were boiled for 10 min before running on 16% Tris-glycine gels. Western blotting was carried out according to Unit protocol, using 3F4 (Merck Inc, N.J., U.S.A) as the primary antibody and goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma A2179) as the secondary antibody.
Substrates were seeded with a 1/100 dilution of vCJD (I4618) 10% brain homogenate in PBS. Each reaction mixture was divided in two prior to PMCA with one half stored at −70 °C as a minus PMCA control. PMCA consisted of 96 cycles of 30 s sonication every 30 min in a Misonix S3000 at 75% power output (Misonix, Farmingdale, NY), reactions were carried out with 40 µl substrate in 200-µl thin-walled PCR tubes at 35 °C.
Samples were digested with 50 µg ml−1 proteinase K (PK) for 1 h at 37 °C. The reaction was stopped by the addition of AEBSF in SDS-loading buffer and samples were boiled for 10 min before running on 16% Tris-glycine gels. Western blotting was carried out according to Unit protocol, using 3F4 (Merck Inc, N.J., U.S.A) as the primary antibody and goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma A2179) as the secondary antibody.
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