Multi-purpose embryo extracts- a freon-free protocol to remove yolk from oocyte extracts.
Source: Veenstra Lab, Radboud Centre for Molecular Life Sciences, the Netherlands
Diluted yolk-depleted embryo extracts:
Yolk can be depleted from whole-embryo lysates by simple centrifugation.
Please note that high salt or ionic detergents (SDS) partially solubilize yolk, which interferes with clearing the extract.
Procedure
-
Collect staged embryos (up to 50 / tube)
-
Remove excess buffer / water
-
Snap freeze in liquid nitrogen / dry ice
-
Store at -80°C
-
Add 4 volume equivalents of room temperature WCE-LS buffer to frozen embryos
NB One embryo corresponds to approx. 1 μl
Final concentration 0.2 embryo equivalent / μl (X. laevis)
- Quick-thaw and homogenize embryos by pipetting up and down (yellow tip), then keep on ice
- Spin in eppendorf centrifuge for 5 mins at max speed at 4ºC
- Quick-freeze (aliquots of) supernatant in liquid nitrogen / dry ice, store at -80°C
Low-salt whole cell extract (WCE-LS) buffer
20 mM Tris pH 7.5
70 mM KCl
1 mM EDTA
10 % Glycerol
5 mM DTT
0.125 % Nonidet P40 / IGEPAL CA-630
Add “Complete” protease inhibitor, or cocktail of leupeptin, pepstatin A aprotinin 1 μg/μl each
Example: Veenstra GJ, Destrée OH, Wolffe AP. Translation of maternal TATA-binding protein mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition. Mol Cell Biol. 1999 Dec;19(12):7972-82. doi: 10.1128/MCB.19.12.7972. PMID: 10567523; PMCID: PMC84882.