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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

PREPARATION OF CHICK EMBRYO EXTRACT

Preparation of Chick Embryo Extract
 
Overview
This protocol describes the production of an extract from several dozen chick embryos.
 
Procedure
1. Tare two 400 ml sterile beakers and chill them on ice along with a third 400 ml sterile beaker.

2. Sterilize approximately 800 ml of cold BSS through a 0.22 um filter.

3. Candle 5 to 6 dozen 12 day old eggs under a tissue culture hood and discard any unfertilized eggs (see Hint #1).

4. Wash the shells of the eggs with 95% Ethanol.

5. Open the egg shells with a pair of flamed tweezers.

6. Reflame the tweezers and open the membrane. Remove the embryo, decapitate it with the tweezers, and place it into a sterile beaker on ice containing about 200 ml of BSS.

7. When all of the embryos are in the beaker, swirl them in the BSS to wash away the blood. Decant the BSS and add another 200 ml of cold BSS.

8. Repeat Step 7 two more times.

9. Lift the embryos with a pair of flamed tweezers and place them into a sterile, empty, tared beaker and determine the mass of the embryos.

10. Add 1 ml of sterile, ice-cold MEM per gram of tissue to the beaker containing the embryos.

11. Transfer the embryos and media to a sterile, pre-chilled Waring blender and homogenize the embryos for 1 min at a setting of 120 on the Variac (medium power).

12. Transfer the homogenate to a chilled sterile beaker and incubate for 1 hr without stirring at 4°C (i.e. in a refrigerator).

13. Dispense the homogenate with a 25 ml pipette into pre-chilled, autoclaved 10 ml Oakridge tubes in an ice bucket in the tissue culture hood.

14. Save the last portion of the extract in a tube at room temperature for several days to check for contamination.

15. Freeze the tubes of embryo extract immediately or centrifuge them at 39,000 rpm (192,000 X g) for 30 min in a SW40 rotor and freeze the decanted supernatant at -20°C.

Solutions
95% (v/v) Ethanol
BSS   1.4 mM Sodium Phosphate Dibasic (Na2HPO4)
1 mM CaCl2
0.15 mM Sodium Phosphate Monobasic (NaH2PO4)
2.7 mM KCl
1 mM MgCl2
135 mM NaCl
 
BioReagents and Chemicals
Sodium Phosphate, Dibasic
Ethanol
Sodium Phosphate, Monobasic
MEM
Magnesium Chloride
Sodium Chloride
Calcium Chloride
Potassium Chloride
 
Protocol Hints
1. Work in a tissue culture laminar flow hood up until weighing the embryos in Step #9.