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.
Yolk Sac Disruption and Purification
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.
Corresponding Organization :
Other organizations : Max Planck Institute of Molecular Cell Biology and Genetics
Protocol cited in 10 other protocols
Variable analysis
- Mechanical stress to disrupt the yolk sac
- Dissolution of the yolk in the deyolking buffer
- Presence of the yolk in the supernatant after low-speed centrifugation
- Deyolking buffer composition (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3)
- Wash buffer composition (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris/Cl pH8.5)
- Shaking time (5 min at 1100 rpm)
- Centrifugation speed (300 g for 30 sec)
- Number of embryos transferred (up to 100 in 200 μl)
- Not specified
- Not specified
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