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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

XENOPUS EGG CSF EXTRACT PREPARATION

Xenopus Egg CSF Extract Preparation
Contributor: The Laboratory of Rebecca Heald in collaboration with John Merlie, Jr. at the University of California, Berkeley
 
Overview
This protocol describes the preparation of Cytostatic Factor (CSF) from frog eggs. CSF can be used in in vitro spindle assembly assays.
 
Procedure
A. Frog injections

1. 3 to 12 days before preparation, inject the adult female Xenopus with 100 Units Pregnant Mare Serum Gonadotrophin.

2. 16 hr before eggs are needed, inject frogs with 500 Units Human Chorionic Gonadotropin.

3. Place each frog in a separate 4 liter container with 2 liter of 1X MMR. Place the containers at 16°C, out of direct light, until egg collection and extract preparation.

B. Preparation Before Starting the Extract Preparation

1. Read over the entire protocol to avoid surprises during the preparation.

2. Check the quality of the laid eggs before preparing reagents and equipment.

3. Get all the solutions ready and place the tubes in a rack

4. Make sure all centrifuges to be used are at 16°C.

5. Make a cut-off Pasteur pipette by nicking the pipette at the neck with a file, snapping it off and flaming the broken end until it is smooth.

C. Extract Preparation

1. If necessary, squeeze the abdomen of the frogs in their 4 liter container of MMR to increase the egg yield.

2. Move the frogs from the 4 liter containers into a bucket containing ddH2O.

3. Check the quality of the eggs that have been recovered. If the eggs are in strings or are white and puffy, they should be discarded using transfer pipettes. If 10% or more of the eggs in a container are like this, all the eggs in that container should be discarded.

4. Collect all the good eggs in a 400 ml beaker.

5. Remove the MMR and add fresh MMR for 5 min. Repeat this washing process until the eggs are clear of debris. All washes from this point on can be done in the 400 ml beaker, swirling after the addition of each solution.

6. Dejelly the eggs in three washes of dejellying solution until the eggs pack down to one-third of their original volume. The packing will happen rapidly after about 5 min of exposure to Cysteine.

7. As soon as the eggs pack to one-third of their volume, pour off the dejellying solution and quickly wash 3 or 4 times with XB solution. After the washes, remove any eggs that are puffy or are lysed.

8. Wash the eggs 3 times in CSF-XB.

9. Wash the eggs 3 times in CSF-XB-LPC. Do not pour off the last wash.

10. Using a cut-off Pasteur pipette, transfer the eggs into 1 ml of CSF-XB-LPC containing 100 μg/ml cytochalasin D in 13 x 51 ultraclear tubes. Keep the eggs completely submerged at all points during the transfer to ultraclear tubes.

11. Load the ultraclear tubes into Starstedt tubes containing 0.5 ml ddH2O.

12. Centrifuge in a clinical centrifuge for 1 min at 200 X g and then increase the centrifugation to 800 X g for 30 sec.

13. Remove any excess buffer and load the tubes into an HB-6 rotor.

14. Centrifuge the tubes at 16,000 X g in the HB-6 rotor for 15 min at 16°C.

15. Remove the tubes from the rotor. Remove the ultraclear tubes from the Starstedt tubes and place them on ice.

16. Puncture the ultraclear tube at the bottom of the clear layer with a needle and syringe. Withdraw all of the clear layer while avoiding all others. Remove the needle from the syringe before expelling its contents into a fresh microcentrifuge tube.

17. Add 0.001 volume of LPC and cytochalasin D stock and 0.025 volume of energy mix. The extract is now ready for use or freezing.

Solutions
10 mg/ml Cytochalasin D (CAUTION! see Hint #1) in DMSO   Store at -20°C in aliquots
CSF-XB   100 mM KCl
0.1 mM CaCl2
10 mM HEPES, pH 7.7
50 mM Sucrose
5 mM EGTA
2 mM MgCl2
LPC   Make up in DMSO (CAUTION! see Hint #1) and store at -20°C in aliquots.
10 mg/ml Leupeptin (CAUTION! see Hint #1)
10 mg/ml Pepstatin (CAUTION! see Hint #1)
10 mg/ml Chymostatin (CAUTION! see Hint #1)
XB   100 mM KCl
0.1 mM CaCl2
10 mM HEPES, pH 7.7
50 mM Sucrose
1 mM MgCl2
Energy Mix   150 mM Creatine Phosphate
Store at -20°C in 100 μl aliquots.
20 mM ATP
20 mM MgCl2
2 mM EGTA
0.5 M EGTA   Dissolve with NaOH pellets in ddH2O and then adjust pH to 7.7.
Filter Sterilize and store in 2 ml aliquots at -20°C.
Dejellying Solution   1X XB Salts
40 mM NaOH
2% (w/v) Cysteine
CSF-XB-LPC   50 μl LPC
50 ml CSF-XB
1 M HEPES, pH 7.7   Sterile filter and store in 5.5 ml aliquots at -20°C
2 M Sucrose   Sterile filter and store in 13 ml aliquots at -20°C
XB Salts (20X)   Sterile filter and store at 4°C
2 mM CaCl2
20 mM MgCl2
2M KCl
MMR   100 mM NaCl
autoclave and store at room temperature
2 mM CaCl2
2 mM KCl
1 mM MgCl2
5 mM HEPES, pH 7.8
0.1 mM EDTA
 
BioReagents and Chemicals
Pregnant Mare Serum Gonadotrophin
Human Chorionic Gonadotropin
Cytochalasin D
HEPES
EDTA
Potassium Chloride
Sodium Chloride
ATP
EGTA
Sucrose
Calcium Chloride
Sodium Hydroxide
Chymostatin
Magnesium Chloride
Pepstatin
Creatine Phosphate
DMSO
Cysteine
Leupeptin
 
Protocol Hints
1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.