Whole embryo protein extraction with freon (Conlon lab)
Protocol submitted by VGP from Conlon lab protocols 
link to protocol document 
Whole Embryo Protein Extraction with Freon
(necessary for removing lipid and yolk)
1) Collect 10 embryos, snap freeze in 1.6 mL microfuge tube with Dry Ice/Ethanol Bath (removing all liquid around embryos). Store @ -80˚C.
2) Add 100 μl lysis buffer while the embryos are still frozen. Keep the proteins on ice at ALL TIMES. I use the following buffer:
- 100 mM NaCl
- 20 mM NaF
- 50 mM Tris, pH 7.5
- 5 mM EDTA
- -store this stock at 4˚C
before use add:
- 1% (v/v) NP40 (IGEPAL)
- 1% (w/v) Na Deoxycholate
- 1:50 EDTA-free Complete Protease Inhibitor (Roche)
3) Lyse embryos with pipetting and vortexing (you can sonicate if necessary).
4) Add 200 μl Freon (1,1,2-Trichlorotrifluoroethane, HPLC Grade, Sigma-Aldrich, Cat. #. 270369- 100 mL)
5) Vortex. Spin @ 4˚C for 15 minutes. Transfer the UPPER PHASE to a new tube. There should be a thin membrane of lipid and pigment between the upper and lower phase.
6) I have found that 15 μL of the protein extract can be run on an SDS-PAGE gel to detect an injected mRNA. If the protein isn’t detectable, you can concentrate it by adding 1 ml Acetone, spinning 20 minutes @ 4˚C, and resuspending in 15 μL lysis buffer.
Daniel Brown – 7-28-2003