. 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 M ammonium sulfate/20 mM acetate buffer (pH ) at room temperature, then luciferase was elute
. 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 M ammonium sulfate/20 mM acetate buffer (pH ) at room temperature, then luciferase was eluted with M ammonium sulfate/20 mM acetate buffer (pH ), and the active fractions were collected. Luciferase fractions that were eluted with ammonium sulfate concentrations lower than 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 M ammonium sulfate, and then adsorbed on a column of Ether-650M ( cm X cm). The adsorbed luciferase was eluted with M lauroylcholine chloride (LCQ/20 mM acetate buffer (pH ), giving about 6 ml of concentrated luciferase solution. Step 3: Size-exclusion chromatography was carried out on a column of Superdex 200 Prep (Pharmacia; cm X 72 cm) with 1 M KC1 LCC/20 mM acetate buffer (pH ) 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 LCC/20 mM acetate buffer (pH ), then adsorbed onto the column. After washing the column with M KC1
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
