By Nathan P. Colowick, Nathan P. Kaplan, Frank Chytil, Donald B. McCormick
The significantly acclaimed laboratory normal, Methods in Enzymology, is without doubt one of the so much hugely revered courses within the box of biochemistry. in view that 1955, every one quantity has been eagerly awaited, usually consulted, and praised through researchers and reviewers alike. The sequence comprises a lot fabric nonetheless proper at the present time - actually an important ebook for researchers in all fields of lifestyles sciences
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Extra resources for Vitamins and Coenzymes Part G
After centrifugation at 105,000 g for 90 min, the supematant is removed, the pellet reextracted and centrifuged as above, and the two supematants combined. Glycerol to a final concentration of 20% is then added to stabilize the enzyme. Step 3. DE-52 Column Chromatography. O mM DTT. O A4 NaCl. From this procedure, as shown in Fig. 2, two peaks of enzyme activity were collected. Each 28 THIAMIN: 40 PHOSPHATES AND 50 55 45 Fraction 151 ANALOGS 60 Number FIG. 3. Elution pattern of Sephacryl S-200 column chromatography.
Airas, And. Biockem. 134, 122 (1983). * R. K. Airas, this series, Vol. 62, p. 267. METHODS IN ENZYMOLOGY, VOL. 122 Copyright D 1986 by Academic Press, Inc. All rights of reproduction in any form reserved. 34 PANTOTHENIC ACID, COENZYME A, AND DERIVATIVES f61 FIG. 1. Standard curve of the pantothenate assay. The reaction mixture (100 ~1) contained 1 mM p-alanine with 3750 counts/set of p-[14C]alanine. Samples of 60 ~1 from the reaction mixture were drawn off at IO-min reaction times. No corrections for the background have been made.
2), in a commercial Waring blender for 4 min at high speed. The suspension is centrifuged for 10 min at 10,000 g and the supernatant recovered by aspiration. Step 2. Microsomal Agglutination. 2 by the addition of 1 M formic acid and the suspension centrifuged for 10 min at 20,000 g. The precipitate is resuspended in -400 ml of deionized water, first by shaking in the original bottles, followed by homogenization in a Waring blender at high speed for 20 sec. 0). Step 3. Solubilization by Treatment with I-Butanol.