Whole embryo protein extraction with freon (Conlon lab)

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Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

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
-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