Phenylmethylsulfonyl fluoride, solution preparation

Highly purified mitochondria and peroxisomes were prepared as described previously. The liver tissue was homogenized in a solution containing 0.25 M sucrose, 1 mM EDTA, 0.1 % ethanol, 5 mM HEPES-KOH (pH 7.4), and 0.2 mM phenylmethylsulfonyl fluoride. The light mitochondrial fraction obtained by differential centrifugation of the homogenate was layered on top of a 12-mL Nycodenz linear gradient (density, 1.15 to 1.25 g/mL) with a 1-mL cushion of... 

After collection and dechorionation at room temperature, washed, dechorio-nated embryos should be quick-frozen in liquid N2 and stored at -70 C. Storage for periods of up to 1 year or longer seems to be without adverse effect. Immediately before fractionation, chilled (4°C) extraction buffer (Buffer E) should be prepared Buffer E contains 5 m Af MgC, 50 mM NaCl, 50 m M Tris-HCl, pH 7.5, 250 mM sucrose, 2.5 mM N-ethylmaleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM L-tosylamide 2-phenyIethyl chloromethyl ketone (TPCK). NEM, PMSF, and TPCK should be included as protease inhibitors. About 20-40 min before fractionation begins, all protease inhibitors are added in solid form to otherwise complete Buffer E in amounts greater or equal to those specified. After addition of protease inhibitors. Buffer E should be stirred continuously at 4°C. Both PMSF and TPCK are incompletely soluble in aqueous solution at the concentrations specified. However, residual undissolved reagents constitute a solid reservoir that becomes completely depleted during embryo fractionation, due to either hydrolysis, protein modification, or both. 

To purify lamins. Drosophila extracts prepared as described above should be supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Lamins from these extracts should be batch adsorbed to antilamin/protein A/sepharose by incubation overnight at 4°C. The column should then be washed extensively with equilibration buffer and purified lamins recovered from the antilamin affinity column with elution buffer. After elution, the solution containing the lamins should be immediately neutralized with Na2HP04 added to a final concentration of 50 mM. Immunoaffinity-purified lamins should be aliquoted, frozen by immersion in liquid nitrogen, and stored at -70°C until use. 

PMSF (100 mM) (phenylmethylsulfonyl fluoride). Prepare the stock in ethanol store at -20°C. The PMSF come out of solution at -20"C, so it must be redissolved just before use (place at 37"C just until it dissolves). 

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