Protocol for making cycling egg extracts from Xenopus laevis

This protocol is based on Andrew W. Murray, Cell cycle extracts. Methods Cell Biol 36, 581-605 (1991).

At least 3 days prior to extract preparation, prime Xenopus laevis female frogs by injecting 100 U per frog pregnant mare serum gonadotropin (PMSG). The day before the preparation, induce ovulation in each frog by injecting 500 U of human chorionic gonadotropin (hCG). All injections are subcutaneous injections into the dorsal lymph sac with a 28-gauge needle.  After injecting hCG, frogs are placed in egg laying buffer (100 mM NaCl, 2 mM KCl, 1 mM MgSO4, 2.5 mM CaCl2, 0.5 mM HEPES-potassium salt, 0.1 mM EDTA-sodium salt; make a 20x stock solution and adjust to pH7.4 using NaOH, store at room temperature; dilute to 1x with frog tank water before using) to collect laid eggs overnight at 18 to 20 °C.  Each frog is isolated in a separate container for egg collection. Eggs are usually collected 16 h to 18 h after hCG injection.

Buffers

1. Extract buffer (XB)

   100 mM KCl

   0.1 mM CaCl2

   1 mM MgCl2

   10 mM HEPES

   50 mM sucrose

   Adjust to pH7.7 with KOH

2. Dejellying buffer

   1x XB salts (100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2)

   2% w/v cysteine, free base

   Adjust to pH7.8 with NaOH

3. 0.2x MMR

   20 mM NaCl

   0.4 mM KCl

   0.2 mM MgCl2

   0.4 mM CaCl2

   0.02 mM EDTA

   1 mM HEPES

   Adjust to pH7.8 with NaOH

4. Activation solution

   Add 5 µl of calcium ionophore A23187 (10 mg/mL stock in DMSO) to 100 mL of 0.2x MMR

5. Protease inhibitor wash buffer

   Dilute leupeptin (10 mg/ml stock in DMSO), pepstatin (10 mg/ml stock in DMSO) and chymostatin (10 mg/ml stock in DMSO) at 1:1000 with 25 ml XB.

6. Crushing buffer

   To 1 ml of protease inhibitor wash buffer, add cytochalasin B (10 mg/ml stock in DMSO) to a final concentration of 100 µg/ml.

Egg extract preparation procedure

The following procedure assumes that about 25 ml of eggs are collected, which is the typical yield for a female frog 16 h after hCG injection.

1. Collect eggs from one frog in a 500 ml glass beaker, and pour off as much as possible the egg laying buffer.

2. Add 100 ml dejellying buffer to eggs, and swirl the beaker periodically to mix. The jelly coat usually comes off in about 5 minutes. There is usually space between eggs due to the presence of the jelly coat, so when the eggs are touching each other, the jelly coat has come off completely. Pour off the dejellying buffer as soon as you confirm that the coat is off and rinse them once with 100 ml of dejellying buffer. Proceed immediately to the next step.

3. Pour off as much dejellying buffer as possible, and then wash the dejellied eggs a few times with a total volume of 300 ml 0.2x MMR. In each wash, decant as much MMR solution as possible, and resuspend eggs with fresh MMR.

4. Pour off as much MMR solution as possible, and add 100 ml of activation solution to activate the eggs. Swirl gently to mix. Activation causes a contraction of the pigmented region of the egg. Eggs with pigmented half facing down previously will also turn and face up after activation. Activated eggs will also become quite sticky and will adhere to each other. Leave the eggs in the activation solution until all of the eggs are activated, which usually happens within 2-4 minutes. This is the most important step, and one must make sure that all the eggs are activated, and then start a timer for a 15-minute room temperature incubation.

5. Pour off the activation solution, and wash the eggs a few times with a total volume of 400 ml XB. Remove irregular eggs using a transfer pipette with the orifice trimmed to be wider than 2 mm.

6. Wash the eggs twice with protease inhibitor wash buffer.

7. Add 1 ml of crushing buffer to an ultracentrifuge tube. Trim the tip of a transfer pipette to create a large slanted opening that allows several eggs to pass through at once. Carefully and slowly transfer the eggs into the ultracentrifuge tube (avoid rupturing the eggs), taking care to minimize the carry-over of XB buffer. Let the eggs settle in the tube. Remove excess buffer on the top of the settled eggs with a Pasteur pipette.

8. Overlay 1-2 ml of Nyosil M25 lubricant on top of the eggs.

9. Centrifuge the tube at room temperature for 60 seconds at 150 x g, and then 30 seconds at 600 x g in a clinical centrifuge. The inert Nyosil M25 lubricant will displace the interstitial XB trapped between the eggs, making the final extract less diluted. At the end of this step, displaced interstitial XB will form a layer on the top, followed by a layer of Nyosil M25, and then by tightly packed eggs. Remove as much as possible the XB and Nyosil M25 layers using a Pasteur pipette. Leave the packed eggs at room temperature until the timer set in step 4 reaches 15 minutes. Then transfer the centrifuge tube to an ice-water bath and incubate for another 15 minutes.

10. Crush the eggs by centrifugation at 16000 x g for 10 minutes at 2 °C in a swinging bucket rotor.

11. After centrifugation, the eggs will have ruptured and separated into several layers from top to bottom: a light yellow lipid layer, a gray or brown cytoplasmic section with a sand colored layer towards the bottom and droplets of Nyosil M25 lubricant near the top, and a solid yolk/pigment layer. Puncture the side of the tube at the bottom of the cytoplasmic section with a 16-gauge needle attached to a 5 ml syringe, making sure the bevel of the needle tip is facing up. Slowly draw the cytoplasmic section into the syringe. This is the crude cytoplasmic extract.

12. Dispense the extract into a 1.5 ml Eppendorf tube, add leupeptin, pepstatin, chymostatin and cytochalasin B to a final concentration of 10 µg/ml each. Mix by inverting the tube a few times.

13. If the extract looks very turbid with a lot of lipid and Nyosil M25 contamination, spin the tube in a table top centrifuge at 16000 x g at 4 °C for 10 minutes. Trim a P-1000 pipette tip with a razor blade to create a wide-bore opening, and transfer the extract to a new tube, leaving behind the darkly pigmented portion at the bottom. If Nyosil M25 contamination is still substantial (it does not affect cycling performance though), transfer the resulting extract to a 400 µl Snap Cap Microcentrifuge Tube (485050 E&K Scientific) and spin at 4 °C for 5-10 minutes in a Beckman Microfuge E with a horizontal rotor. Cut off the top section that contains the Nyosil M25 droplets and cut off the bottom tip that contains residual darkly pigmented particles with a clean razor blade. Recover the remaining extract, and hold on ice until use.

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