Recipies deyolking buffer (DYB) (50 ml)
49.43 ml sterile water
550 µl 5M NaCl (55 mM)
90 µl 1M KCl (1.8 mM)
62.5 µl 1M NaHCO
3
(1.25 mM) add fresh leupeptin and pefabloc at the appropriate concentrations
*optional: add 2.7 mM CaCl
2
(135 µl of 1M CaCl
2
) to keep cells adhering to one another during deyolking wash buffer (WB) (50 ml)
48.1 ml sterile water
1.1 ml 5M NaCl (110 mM)
175 µl 1M KCl
500 µl 1M tris pH 8.5 (10 mM)
135 µl 1M CaCl
2
(2.7 mM)
Procedure
1. Dechorionate. If you are making lysates from pre-TB stage embryos, it might be a good idea to dechorionate the embryos using Pronase E before or soon after injection.
Don’t use Pronase E immediately before making the lysate, as this might lead to protein degradation. Wash out the pronase E well before continuing.
2. Add 1 ml of DYB to embryos and disrupt the yolk by pipetting repeatedly with 1 ml pipette. Not too much, just enough to break the yolk sack
3. Vortex at 1100 rpm (~4 on our vortexers) for 30 sec to dissolve the yolk
4. Pellet the cells by centrifuging at 2000-2500 rpm for 30 sec
5. Remove the supernatant and add 1 ml of WB
6. Repeat steps 3 - 4
7. Remove supernatant. Lyse cells in 1X sample loading buffer (2.5% Bmercaptoethanol). Ad d 3 µl loading buffer per embryo