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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

CHICK EMBRYO EXTRACT

Chick Embryo Extract Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of David Anderson at the California Institute of Technology
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Chick Embryo Extract is an essential source of growth factors for growing rat neural crest stem cells and other types of neural explants.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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 Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Potassium Phosphate, Monobasic L-Valine Calcium Chloride L-Tyrosine Sodium Bicarbonate Inositol Sodium Phosphate Eggs, Chicken Fertilized Hyaluronidase Type IV-S, from bovine testes Phenol Red Thiamine-HCl Ethanol L-Tryptophan L-Threonine Sodium Phosphate, Dibasic Potassium Chloride Sodium Chloride L-Phenylalanine L-Methionine Cysteine-HCl L-Glutamine Riboflavin L-Leucine L-Isoleucine Pyridoxal Niacinamide Choline Chloride D-Ca Pantothenate Lysine-HCl L-Histidine-HCl L-Arginine-HCl Folic Acid D-Glucose Magnesium Sulfate
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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.