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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PURIFICATION: EXTRACT FROM CELLS
CHICK EMBRYO EXTRACT
Chick Embryo Extract
Contributor: The Laboratory of David Anderson at the California Institute of Technology Overview
Chick Embryo Extract is an essential source of growth factors for growing rat neural crest stem cells and other types of neural explants. Procedure
1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
2. Wash the surface of the eggshells carefully with 70% Ethanol.
3. Crack open the egg. Dissect out the embryos and place them in MEM at 4°C. Use approximately 7 ml of MEM for every 3 dissected embryos.
4. Macerate approximately 10 embryos at a time by passing them through a 30 ml syringe into a 50 ml sterile centrifuge tube (see Hint #2).
5. Add an equal volume of MEM at 4°C to the tube of embryos. Incubate with rotary shaking action for 45 min at 4°C.
6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix.
7. Centrifuge the mix at 30,000 X g for 6 hours at 4°C (16,000 rpm for 6 hours using a Sorvall™ SS34 rotor).
8. Filter the supernatant through a 0.45 μm filter and then through a 0.22 μm filter using sterile technique.
9. Aliquot and store at -80°C.
Hyaluronidase Store at -80°C
Filter through a 0.2 micron filter
10 mg/ml Hyaluronidase (800 Units/mg), Type IV-S, from bovine testes
Aliquot into 100 μl aliquots
Prepare in D-PBS
MEM (1X) 0.01 mM Inositol
1.8 mM CaCl2
0.4 mM L-Isoleucine
0.1 mM L-Methionine
0.4 mM L-Valine
0.05 mM L-Tryptophan
26.2 mM Sodium Bicarbonate (NaHCO3)
5.3 mM KCl
0.4 mM L-Leucine
117.24 mM NaCl
0.0002 mM Riboflavin
0.008 mM Niacinamide
0.1 mM Cysteine-HCl
0.005 mM Pyridoxal
0.002 mM Folic Acid
0.4 mM L-Threonine
0.002 mM D-Ca Pantothenate
1 mM Sodium Phosphate (NaH2PO4- H2O)
0.003 mM Thiamine-HCl
2 mM L-Glutamine
0.2 mM L-Phenylalanine
0.025 mM Phenol Red
0.4 mM Lysine-HCl
0.007 mM Choline Chloride
0.2 mM L-Tyrosine
0.2 mM L-Histidine-HCl
0.6 mM L-Arginine-HCl
5.55 mM D-Glucose
0.814 mM MgSO4
70% (v/v) Ethanol D-PBS (1X) 0.50 mM MgCl2
1.47 mM Potassium Phosphate Monobasic (KH2PO4)
0.90 mM CaCl2
Dulbecco's PBS containing Ca and Mg
138.00 mM NaCl
8.10 mM Sodium Phosphate Dibasic (Na2HPO4-7H2O)
2.67 mM KCl
BioReagents and Chemicals
Potassium Phosphate, Monobasic
Eggs, Chicken Fertilized
Hyaluronidase Type IV-S, from bovine testes
Sodium Phosphate, Dibasic
1. The length of time that the eggs are incubated depends on the age of the eggs when they arrive at the lab. Normally, eggs that are ordered arrive within 2 to 3 days of being laid. Most universities and educational institutions have agreements with neighboring or local farms. Please check with your animal facility at your institution for a source for fertilized chicken eggs.
2. This should produce approximately 25 ml of volume.