. Le bulletin biologique. Biologie ; zoologie ; biologie ; biologie marine. La LUCIFÉRASE PERIPHYLLA STOCKÉES DANS OVAIRE 341. 50 60 70 80 90 100 110 volume d'élution (ml) Figure 1. Un exemple de la troisième étape de filtration sur gel Superdex 200 Prép. Les courbes d'élution sont indiquées pour l'activité de luminescence (ligne solide) et la valeur de A280 nm i Cm (ligne pointillée). A, B, et C sont les sommets du luciferases A, B et C respectivement. Les fractions constituant chacun de ces pics ont été combinées pour une purification supplémentaire. avec le sulfate d'ammonium 2,2 M/20 mM Tampon acétate (pH 4,8) à température ambiante, puis éluer était luciférase

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. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. PERIPHYLLA LUCIFERASE STORED IN OVARY 341. 50 60 70 80 90 100 110 Elution volume (ml) Figure 1. An example of the third-step gel filtration on Superdex 200 Prep. Elution curves are shown for luminescence activity (solid line) and the value of A280 nm i Cm (dashed line). A, B, and C are the peaks of luciferases A, B. and C. respectively. The fractions constituting each of these peaks were combined for further purification. with 2.2 M ammonium sulfate/20 mM acetate buffer (pH 4.8) at room temperature, then luciferase was eluted with 1.8 M ammonium sulfate/20 mM acetate buffer (pH 4.8), and the active fractions were collected. Luciferase fractions that were eluted with ammonium sulfate concentrations lower than 1.8 M were not used in this study. Extracts 3 and 4 were chromatographed on the Ether-650M column in the same manner. All of the active fractions were combined, made up to 2.4 M ammonium sulfate, and then adsorbed on a column of Ether-650M (1.5 cm X 3.5 cm). The adsorbed luciferase was eluted with 0.5 M K.Cl/0.01% lauroylcholine chloride (LCQ/20 mM acetate buffer (pH 4.8), giving about 6 ml of concentrated luciferase solution. Step 3: Size-exclusion chromatography was carried out on a column of Superdex 200 Prep (Pharmacia; 1.5 cm X 72 cm) with 1 M KC1/0.01% LCC/20 mM acetate buffer (pH 4.8) as the eluent. On each run. 3 ml of the sample were injected and the effluent was collected in 2-ml fractions. The fractions were separated into 3 groups—luciferases A, B and C (see Fig. 1)—according to their elution volume, and all the fractions from the same group were combined. Step 4: Cation-exchange chromatography was carried out on a column of SP Sepharose High Performance (Pharma- cia; 1 cm X 6 cm) at room temperature. The eluate from the third step was diluted with two volumes of 0.01% LCC/20 mM acetate buffer (pH 5.5), then adsorbed onto the column. After washing the column with 0.5 M KC1/0.01

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• bookauthorlilliefrankrat
• booksubjectbiology
• booksubjectzoology