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Preparation of high speed supernatants HSS of Xenopus egg extract
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Day 1 -
What you need before getting started:
- 2 syringes of 1 mL with 2 small needles and 2 mL aliquots of PMSG
- 2 small Plexiglas tanks
Take some water from the big tank in the two Plexiglas tank. Select 4 frogs and put them in the small tanks (2 per tank). Try to choose different colors to recognize which frogs you have injected.
Inject 0.5 mL of PMSG (50 units) (pregnant mare serum gonadotropin) per frog in the dorsal lymph sac (pregnant mare serum gonadotropin)
Put the frogs in a new big tank
Feed on regular schedule
Day 5 to day 12 - Induction of ovulation.
What you need before getting started :
- 2 syringes of 1 mL with 2 small needles and 2.2 mL aliquots of HCG
- 2 small Plexiglas tanks with 2L MMR precooled to 16°c in each
The 2 x 2 frogs are placed in 2 containers containing 2L of MMR pre-cooled to 16°c
Inject 0.6 mL HCG 150(units) (Human Chorionic Gonadotropin)
Keep the frogs in the 2 small Plexiglas tanks all night
Day 6 –
What you need before getting started:
- 10 x stock MMR + graduated cylinder take the 10 x stock
- 4 beakers to stock the eggs
- 1 big beaker to pull the eggs
- transfer pipettes
12 to 14 hours later, you have the eggs.
Eggs are collected 16-20 hr after HCG injection
Verify the quality of the eggs in the Plexiglas tanks.
Take each frog and remove eggs by tightening and slipping your hands on the frog’s abdomen.
Collect the eggs in a beaker before pulling them to see if there are good or bad eggs.
Put the frogs in their big tank and put a note with the day’s date to know not use theses frogs until 4 months from now
Pull good eggs and remove bad eggs with a transfer pipette.
Eggs are washed with MMR to remove frog skin and detritus (5 times)
A 4 frog prep, collection, sorting and washing should take no longer than 45 min.
Go back to the lab with your eggs.
Making HSS
What you need:
- dejellying solution 250 mL per frog
- transfer pipettes
- CSF-XB buffer 200 mL for washes
- CSF-XB (without sucrose) 20 mL per frog for dilution of extract
- seton centrifuge tubes U7030 (12ml capacity)
- Protease inhibitors
- CSF Energy mix approximately 200 microL aliquots per frog
Remove as much MMR as possible.
Add 4 volumes of dejellying solution and swirl gently for 5 min
The supernatant is decanted carefully and the eggs are rinsed once more with half the original volume of dejellying solution. Repeat until dejellying is complete: when eggs pack as tight spheres without any visible separation caused by their jelly coats.
Before  After 
Dejellied eggs are washed extensively in CSF-XB buffer, ~10 packed dejellied egg volumes each wash. From this point on work as quickly as possible as the eggs are less stable without their jelly coat.
A final wash is conducted with a small volume (equal to eggs) of CSF-XB containing protease inhibitors
Damaged eggs after each wash are discarded using a transfer pipette.
For the subsequent centrifugation steps, use seton centrifuge tubes U7030 (12ml capacity) (Denville Scientific, INC) which allow perforation with a needle and are suitable for a rotor that can reach 10,000 rpm
After the final wash of the eggs with CSF-XB, pour off as much buffer as possible, and transfer the eggs carefully until each centrifuge tube is full. Use a transfer pipette for small volumes.
After transferring the eggs, excess CSF-XB is removed, eggs are spun at 1000 rpm in SW 41 Ti rotor for 1 min @ 4?C to displace interstitial buffer trapped between them, and the excess buffer from the top of the tube is removed again, thereby decreasing the dilution of the cytoplasm in the final extract.
Eggs are then crushed by spinning at 10,000 rpm in SW 41 Ti for 10 min at 4°C. After this step, all handling is done at 4°, and extracts are kept on ice.
After centrifugation, eggs will have ruptured and separated into three major layers of lipid, cytoplasm, and yolk going from top to bottom, with other minor layers at the interfaces.
Cytoplasmic extract is collected using a syringe needle by puncturing the side of the tube at the bottom of the middle layer (see arrow) and very slowly drawing the cytoplasm while avoiding the interfaces. (note: Carefully rotating the needle while puncturing the tube can facilitate perforation)
Protease inhibitors are added to a final concentration of 10 µg/ml (each), energy mix for CSF extracts (20x CSF-energy mix) is added to a final concentration of 1x, and the extract is mixed gently by flicking or inverting.
This is LSS or crude extract.
Making High-Speed Supernatant
Crude cytoplasmic extracts are very viscous and, therefore, must be diluted for efficient sedimentation during high-speed ultracentrifugation.
The crude extract is diluted 5-fold in CSF-XB (without sucrose) with 1 mM DTT. The dilute extract is then transferred to seton centrifuge tubes U7030, 0.5 ml of mineral oil (Sigma M-5904) is layered on top, and the extract is centrifuged at 30,000 rpm (~100,000 x g) for 3 h at 4° in a SW41 Ti. Light membranes that do not pellet during high-speed centrifugation will partition into the mineral oil layer and are removed after centrifugation by aspirating the top layer carefully (the rotor and tubes should be handled with care to avoid mixing). A small amount of contamination with light membranes is unavoidable and does not adversely affect the quality of the HSS. The HSS is carefully recovered by pipetting without disturbing the pellet and is re-spun under the same conditions to further deplete endogenous membranes.
Snap freeze the recovered HSS directly in aliquots in liquid nitrogen
Reagents
CSF-XB
100 mM KCl
2 mM MgCl2
10 mM HEPES, pH 7.7
50 mM sucrose
5 mM EGTA
Prepare 200 mL per frog for washes + 20 mL without sucrose per frog for dilution of extract
Prepare 400 mL per frog
Prepared fresh from :
- 20x XB salt (2 M KCl, 20 mM MgCl2, 2 mM CaCl2), sterile filtered and stored at 4°c
- 1.5M sucrose, sterile filtered and stored in aliquot at -20°c
- 1M Hepes, titrated with KOH to yield a pH of 7.7 when diluted to 10 mM, sterile filtered and stored in aliquots at -20°c
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MMR
100 mM NaCl
2 mM KCl
1 mM MgCl2
2 mM CaCl2
0.1 mM EDTA
5 mM HEPES, pH 7.8
A 10x stock is prepared, titrated with NaOH to pH 7.8, autoclaved, and stored at room temperature.
Prepare 3L per frog
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Dejellying solution
1x XB salt (100mM KCl, 1 mM MgCl2, 0.1 mM CaCl2)
2% W/v cystein, free base
<<< !!!!!! >>> Made up within 1 H of use and titrated to pH 7.8 with NaOH ( 4.5 mL of 1 M NaOH per 2 g of cystein yields the desired pH)
Prepare 250 mL per frog
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CSF Energy mix
150 mM creatine phosphate
20 mM ATP, pH 7.4
2 mM EGTA
20 mM MgCl2
Stored in aliquots at -20°c
Prepare 200 microL per frog
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Protease inhibitor
Mixture of leupeptin , chymostatin, and pepstain dissolved to a final concentration of 10 mg/mL each in DMSO and stored at -20°c
Prepare 32 microL per frog
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PMSG
100 U/mL PMSG made in water and stored at -20°c
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HCG
250 U/mL HCG in water and stored at 4°c (<<<good for at least 2 weeks !!!!!>>>)
We also have frozen aliquots at –80? (see freezer map)
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Oil
Mineral oil (SIGMA) M-5310
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