Journal: Neurochemical research
Article Title: Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A
Figure Lengend Snippet: Ethanol Precipitation Dissociates the Catalytic Subunit from the Regulatory Subunits of PP2A. The effect of ethanol precipitation on the molecular form of PP2A was determined following size exclusion chromatography in Tris–EDTA buffer containing 10 mM β -mercaptoethanol and 100 mM NaCl on a Sephacryl 300HR column of the crude S100 fraction (3.0 mg protein) and S100 fraction following ethanol precipitation, extraction, and ammonium sulfate precipitation to form the EtOH-AS65 fraction (0.15 mg protein). PP2Ac in the fractions was determined by measuring the phosphatase activity toward a phosphothreonine peptide. Phosphatase activity is reported as nmol Pi produced during a 10 min incubation period per 225 μL of the column fractions. γ -Globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) were employed as molecular weight markers
Article Snippet: For each S100 fraction prepared, one whole brain was partially thawed and homogenized in a glass-Teflon homogenizer in 15 mL of Tris–EDTA buffer (50 mM Tris, 1 mM EDTA, 1 mM benzamidine, pH 7.4) to which was added 50 μL of mammalian protease inhibitor cocktail (Sigma Chemical) per brain.
Techniques: Ethanol Precipitation, Size-exclusion Chromatography, Activity Assay, Produced, Incubation, Molecular Weight