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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

PREPARATION OF EMBRYONIC EXTRACT FROM CHICKEN EMBRYOS

Preparation of Embryonic Extract from Chicken Embryos
 
Overview
This protocol describes the preparation of a cellular extract from chick embryos. The embryonic tissue is homogenized and then lysed by repeated freeze/thaw cycles. The lysate is centrifuged and filtered to generate a clarified extract.Use aseptic technique for this protocol, and sterilize all instruments before using protocol.
 
Procedure
1. Spray a dozen 12-day-old fertilized chicken eggs twice with 70% Ethanol.

2. Wipe the eggs with 95% Ethanol thoroughly and let eggs dry completely.

3. Collect 9 to 12 chick embryos in a sterile 100 mm petri dish on ice. Use large scissors, coarse forceps, and a pair of fine forceps for manipulation of the embryos.

4. Cut off the embryo heads and discard, using a pair of fine scissors.

5. Slit embryos ventrally and pierce the heart three to four times to drain embryo blood, using a pair of fine scissors.

6. Cut embryo legs off and discard.

7. Transfer dissected embryos to the second sterile 100 mm petri dish on ice, using a second pair of forceps. Clean out the first dish.

8. Chop each dissected embryo 4 to 5 times with the second pair of fine scissors.

9. Divide Saline G Solution into three 250 ml beakers (approximately 75 ml into each).

10. Spread a square piece of sterile cheesecloth in the bottom of the cleaned first petri dish and transfer chopped embryos onto the cheesecloth.

11. Wrap embryos in cheesecloth and sequentially dip the embryos in each beaker for a few seconds to wash the embryos. After the last dip, squeeze the sack of embryos with the flat side of a pair of forceps against the side of the beaker to drain excess liquid.

12. Transfer the embryonic tissue into a 50 ml sterile beaker sitting on ice. Use a pair of forceps to manipulate solid pieces.

13. Homogenize the tissues with a Brinkmann Polytron (Brinkmann PTA 10TS generator), until individual pieces are no longer visible. Keep the beaker on ice (see Hint #1). Homogenize for 10 to 15 strokes.

14. Transfer no more than 25 ml of the homogenate to a sterile 50 ml conical centrifuge tube. Note the volume of the homogenate.

15. Dilute the homogenate with an equal volume of Hank's Balanced Salt Solution. Use some of the Hank's Balanced Salt Solution to rinse the homogenizer instruments to collect any remaining homogenate before diluting the homogenate. Mix the tube by shaking.

16. Freeze the cell suspension at -70°C for at least an hour to lyse the cells (see Hint #2).

17. After freezing, thaw the tube in cold water until there is a minimal ice pellet left.

18. Repeat the freeze/thaw cycle by repeating Steps #16 and #17.

19. Pellet cell debris by centrifuging the lysate in a Beckman 50.2 Ti rotor at 33,000 rpm (99,000 X g) for 1 hour at 4°C.

20. Pipette the supernatant immediately into a test tube (see Hint #2).

21. Assemble all filters before starting the filtration. Filter no more than 25 ml of homogenate through the prefilters at a time. Filter in the following order:

a. Once through a Millipore thick prefilter type AP

b. Once through a Millipore thin prefilter type AW

c. Two to 4 times through a Millipore 0.8um filter type AA

Handle filters with forceps and collect filtrate in ice-cold 50 ml sterile flasks. Open the holder over the flask to collect residual fluid.

22. Filter the extract through a Cameo IV 0.22 μm filter. Collect filtrate in two 15 ml conical tubes.

23. Store the filtered extract at -20°C. Discard the extract after approximately 2 weeks (see Hint #4).

Solutions
Hank's Balanced Salt Solution   0.6 mM MgSO4
0.4 mM KH2PO4
5.4 mM KCl
1.3 mM CaCl2
pH 7.4
0.5 mM MgCl2
4.2 mM NaHCO3
0.3 mM Na2HPO4
5.6 mM Glucose
137 mM NaCl
Saline G Solution   5.36 mM KCl
0.62 mM MgSO4
1.1 mM Potassium Phosphate Monobasic (KH2PO4)
6 mM Glucose
1.08 mM Sodium Phosphate Dibasic (Na2HPO4)
0.11 mM CaCl2
137 mM NaCl
0.0005% (w/v) Phenol Red
70% (v/v) Ethanol   in a spray bottle
95% (v/v) Ethanol
 
BioReagents and Chemicals
Magnesium Chloride
Liquid nitrogen
Magnesium Sulfate
Potassium Phosphate, Monobasic
Phenol Red
Sodium Phosphate, Dibasic
Potassium Chloride
Calcium Chloride
Sodium Chloride
Ethanol
Sodium Bicarbonate
Glucose
 
Protocol Hints
1. Alternatively, a Dounce homogenizer with the coarse fitting pestle can be used.

2. The suspension may be left overnight in the -70°C freezer if desired.

3. Some turbid material near the top of the supernatant may be included.

4. For longer term storage, place extract at -70°C or below. Extract can be stored indefinitely in Liquid Nitrogen.