A New Method for the Purification of Nuclei from Artemia
Yolk platelets outnumber nuclei about 1400 to 1. Since the densities of these two organelles are comparable, it has been difficult to obtain nuclear preparations free of contaminating yolk platelets using differential centrifugation. We report here on a simple and reliable method for obtaining Artemia nuclei free of yolk platelets using NaCl-Percoll gradients. Free swimming nauplii were collected after 24 hrs. of culture and homogenized in 0.01 M Tris-HCl, 0.15 M NaCl, 0.03 M CaCl2, 0.024 M EDTA, 0.5% Triton X-100, pH 8.0 (SCE). All centrifugations were performed in a Sorvall GSA-600 rotor. Shells and cellular debris were removed by a “quick spin”. The “quick spin” entails allowing the rotor to reach 1000 rpm followed by immediate deceleration. Nuclei were pelleted from the supernatant by centrifugation at 2500 rpm for 10 minutes. This centrifugation was repeated a second time. The nuclear pellet was next resuspended in SCE buffer and centrifuged at 3000 rpm for 10 minutes. Subsequently, the nuclei were resuspended in SCE not containing detergent, layered onto 0.15 M NaCl-Percoll, and centrifuged at 12,500 rpm for 15 minutes. Nuclei, which form a discrete layer just below the surface, were removed with a Pasteur pipette and washed with SCE to remove the Percoll. Microscopic examination revealed isolated, intact nuclei in a field free of yolk platelets. The preparation is free of lactic dehydrogenase and isocitric dehydrogenase enzyme activities and incorporates 3H-uridine triphosphate into trichloroacetic acid precipitable material linearly over a two hour incubation period.