The protein pellet was then dissolved in HENS buffer (1% SDS in HEN buffer; 0.1 mL of HENS buffer per mg of protein in the initial protein sample).
Fecal extraction buffer (0.25 M Tris, 0.25 M citric acid, 0.025 M CaCl2, 2.5 M urea, and 1.25% bovine serum albumin) was added at the ratio of 49 μL buffer per mg of stool to produce a dilution factor of 1 : 50.
A negative control without ascorbate was performed on Prnp+/+ samples to check the efficiency of thiols blocking with S-methyl thiomethanesulfonate. Ascorbate was removed by acetone precipitation and the protein pellet was resuspended in 100 μl of HENS buffer per mg of starting proteins.
DNA was isolated from brain using the Gentra PUREGENE DNA purification kit (QIAGEN Benelux B.V., Venlo, The Netherlands) according to protocol with the exception that all buffer amounts per mg of brain tissue were doubled due to the high protein and fat content of brain material.
S-methyl thiomethanesulfonate was removed by acetone precipitation and the protein pellet was resuspended in 100 μl of HENS buffer (HEN+1%SDS) per mg of starting proteins.
The frozen samples were defrosted over ice, weighed, cut into pieces of ~1 mm, and then homogenised in 1 mL of buffer per 1 mg of tissue.
An extraction buffer (4 % SDS, 2%% β-mercaptoethanol, 2 mM PMSF, 100 mM TrisHCl, pH 8,8) was added in the proportion of 10 μl of extraction buffer per 1 mg of powder mass.
Frozen lung tissue was thawed at room temperature, then placed in 1X phosphate buffered saline (PBS) with 0.5 mM EDTA according to weight (10 μL of buffer per 1 mg of tissue).
Frozen villous samples were homogenized in ice-cold lysis buffer (1 mL of buffer per 100 mg of tissue) containing 20 mmol/L Tris, pH 7.4, 1 mmol/L EGTA, 0.01% Triton X-100, 1 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 10 mmol/L β-glycerol phosphate, 50 mmol/L sodium fluoride, and a complete mini protease inhibitor cocktail (Roche Diagnostics, East Sussex, UK).
Drosophila embryo extracts were produced from dechorionated 0 6 hr wild-type embryos by homogenising in DXB buffer as described (Bullock et al., 2006) using 100 μl of DXB buffer per 50 mg of embryos.
The cell pellets were washed with 25 mM sodium phosphate buffer, pH 7.0 and were subsequently resuspended in 1 mL of the same buffer per 100 mg of cells (wet weight).
