By Nathan P. Colowick, Nathan P. Kaplan, John H. Law, Hans C. Rilling
The significantly acclaimed laboratory general, Methods in Enzymology, is likely one of the so much hugely revered guides within the box of biochemistry. for the reason that 1955, each one quantity has been eagerly awaited, usually consulted, and praised through researchers and reviewers alike. The sequence comprises a lot fabric nonetheless suitable at the present time - actually a necessary e-book for researchers in all fields of lifestyles sciences.
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Additional resources for Steroids and Isoprenoids, Part B, Volume 111 (Methods in Enzymology)
The acidified aliquot is taken to dryness at 95 ° in an aluminum heating block (Lab-Line 2073). 3 ml) is added to dissolve the dried nonvolatile material. After addition of 3 ml of liquid scintillator, the vials are capped and mounted in 25 mm diameter vial holders (Kimble 74700). The nonvolatile 14C radioactivity (due to [14C]HMG-CoA) is measured by liquid scintillation counting and corrected for any nonvolatile Jac radioactivity in [14C]acetyl-CoA (determined from a control in which enzyme is omitted).
One unit of plastid HMG-CoA reductase catalyzes the formation of 1 nmol of mevalonic acid per hour under the above assay conditions. Protein Assay. The modified Lowry procedure described by Peterson 18 is used (for comments, see Method 1, microsomal reductase). Assay Method 2 (Plastid Reductase) For general comments about this microassay procedure, see Method 2, microsomal reductase. Reagents Refer to Method I, plastid reductase, for homogenizing medium; rupture medium; suspension medium; NADPH-generating system; K-phosphate; and dithiothreitol RS-[3-14C]HMG-CoA, 50,000 dpm/nmol (for synthesis, see Method 1, microsomal reductase) See Method 2, microsomal reductase for thin layer plates, diethylether, acetone See Method 1, microsomal reductase for HCI, MVA-lac, scintillation liquid 38 EARLY STAGES IN ISOPRENOID BIOSYNTHESIS  Procedure.
Homogeneous suspensions are readily achieved by initially homogenizing (glass rod) the pellet in the residual liquid, then adding an aliquot ( - 5 0 /xl) of suspension medium, mixing again, then adding the additional suspension medium. Before making to the final volume the homogeneity is checked using a glass micropipet (auto-zero, constriction-type); any visible particulate matter remaining is readily dispersed by 2 or 3 passes through this pipet. 19 W. D. L o o m i s and J. Battaile, Phytochemistry5, 423 (1966).