View source for Whole embryo protein extraction with freon (Conlon lab)

Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot]


link to protocol document [http://www.unc.edu/~fconlon/Protocols/Whole%20Embryo%20Protein%20Extraction%20with%20Freon.doc]


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