Inhibitors continued storage

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. 

The concentrate storage tanks for slurry pipelines are essentially large tanks with agitators (Figure 12-5). A smaU pump test loop near these tanks is used to test the concentrate before feeding it to the pipeline pumps. Feed is essentially from the thickeners, but continuous agitation in the tank and addition of viscosity control agents, corrosion inhibitors, and even some dilution water are part of the process. 

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