The principle behind the following method is to disrupt the yolk sac by mechanical stress. Subsequently, the yolk is dissolved in an appropriate buffer so that it stays in the supernatant during low speed centrifugation.
Embryos were transferred from the agar coated dish to a 1.5 ml tube filled with 1 ml deyolking buffer (1/2 Ginzburg Fish Ringer [1 ] without Calcium: 55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3) by pipetting with a narrow tip so that the yolk sac is disrupted (200 μl tip, Sarstedt 70.760.502). Up to 100 embryos can be transferred in a 200 μl volume. The embryos were shaken for 5 min at 1100 rpm to dissolve the yolk (Thermomixer, Eppendorf). Cells were pelleted at 300 g for 30 sec and the supernatant discarded. Care was taken not to disrupt the soft cell pellet. Optionally two additional wash steps were performed by adding 1 ml of wash buffer (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris/Cl pH8.5), shaking 2 min at 1100 rpm and pelletting the cells as before. The additional wash steps further decreased the yolk and are recommended for 2D gel electrophoresis. The samples were frozen in liquid nitrogen or processed directly for Western blotting or 2D gel electrophoresis.
Embryos were transferred from the agar coated dish to a 1.5 ml tube filled with 1 ml deyolking buffer (1/2 Ginzburg Fish Ringer [1 ] without Calcium: 55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3) by pipetting with a narrow tip so that the yolk sac is disrupted (200 μl tip, Sarstedt 70.760.502). Up to 100 embryos can be transferred in a 200 μl volume. The embryos were shaken for 5 min at 1100 rpm to dissolve the yolk (Thermomixer, Eppendorf). Cells were pelleted at 300 g for 30 sec and the supernatant discarded. Care was taken not to disrupt the soft cell pellet. Optionally two additional wash steps were performed by adding 1 ml of wash buffer (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris/Cl pH8.5), shaking 2 min at 1100 rpm and pelletting the cells as before. The additional wash steps further decreased the yolk and are recommended for 2D gel electrophoresis. The samples were frozen in liquid nitrogen or processed directly for Western blotting or 2D gel electrophoresis.
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