. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 246 O. SHIMOMURA AND P R. FLOOD 015 e c o CO CM c (0 < 0 10 005 000. 30 - 20 3. 3 10 - 0 Elution volume (ml) Figure 1. The last step in purification of PeriphylUi luciferase-L on a column of Superdex 200 Prep (1 cm X 28 cm) with 10 mM acetate buffer, pH containing 1 M KCl. Eluent fractions between ml and 18 ml were pooled and used as the purified luciferase. Solid line: absorbance measured with a 1-cm light-path cuvette; dotted line: luminescence activity measured at a photometer sensitivity of 3 X 10" quan
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 246 O. SHIMOMURA AND P R. FLOOD 015 e c o CO CM c (0 < 0 10 005 000. 30 - 20 3. 3 10 - 0 Elution volume (ml) Figure 1. The last step in purification of PeriphylUi luciferase-L on a column of Superdex 200 Prep (1 cm X 28 cm) with 10 mM acetate buffer, pH containing 1 M KCl. Eluent fractions between ml and 18 ml were pooled and used as the purified luciferase. Solid line: absorbance measured with a 1-cm light-path cuvette; dotted line: luminescence activity measured at a photometer sensitivity of 3 X 10" centrifuged at 20,000 X g for 20 min. The supernatant was discarded. The precipitate was mixed with 5 ml of 10 mM Tris-HCl buffer, pH , containing 2 M guanidine hydrochloride, 1 M KCl and 1 % glucose, then sonicated briefly. The mixture, containing a total activity of , was centrifuged at x g for 20 min. The supernatant contained solubilized luciferase (70,000 ). and the precipitate contained the luciferase that remained insoluble (about ; not used in the present study). Thus, we estimated that the activity of the insoluble form of luciferase had decreased to about 50% by solubilization. One-tifth of the supernatant (1 ml; ) was purified by gel filtration on a column of Superose 6 prep grade (Pharmacia: 1 cm x 19 cm) in 10 mM Tris buffer. pH containing 2 M guanidine hydrochloride. 1 M KCl and glucose. The elution curve is shown in Figure 2. The gel filtration was repeated four more times to purify the rest of the supernatant. Results and Discussion Purification o/Periphylla luciferases The luciferase of the lappets (luciferase-L) could easily be extracted with neutral saline solutions. The luciferase activity was sufficiently stable during purification in neu- tral and acidic media (even at pH and below) at 0°C. but was drastically reduced by dilution with water— by a decrease in the ionic strength.
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