[Xenopus] Freon-free protocol for clearing yolk from Xenopus egg extracts:

[xenbase]

Our collective thanks to Gert Veenstra for this protocol!


Multi-purpose embryo extracts (a freon-free protocol)


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.
PMID: 10567523; PMCID: PMC84882.
doi:10.1128/MCB.19.12.7972.


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