Cycling extracts, sara's protocol (Stukenberg lab)

Protocol submitted by VGP from Stukenberg lab protocols [1]

link to protocol page [2]

Sara's Cycling Xenopus Extracts

To prepare: 5 days before the experiment, prime female frogs with 50 IU of PMSG.

The night before the experiment, induce ovulation by injecting the primed frogs with 500 IU of HCG. Place each frog in 2L of 1X MMR overnight at 18°C.

In the morning, collect laid eggs. Place the frogs back into 1X MMR and continue to collect eggs.

Once you have sufficient amounts of eggs, wash the eggs in 1X MMR to remove debris and bad eggs.

Remove as much MMR as possible and de-jelly the eggs with 2% cysteine solution prepared in 1X XB salts. Swirl eggs occasionally and de-jelly until the eggs pack tightly. Pour off de-jelly, and rinse briefly with fresh de-jelly solution.

Wash the eggs into 1X MMR. Activate the eggs by addition of 2μg/mL A23187, a calcium ionophore. (To 10 mL MMR, add 1μL ionophore). Watch for activation (takes about 10 minutes). Upon activation, wash out ionophore liberally with MMR. Place the eggs at 18°C for 50 minutes.

Wash the eggs 4X with XB, and 2X with XB+protease inhibitors (10μg/mL each leupeptin, pepstatin and chymostatin (LPC)). Load the eggs gently into ultraclear centrifuge tubes containing 1 mL XB+PIs and 100μg/mL cytochalasin B or D.

Pack the eggs by spinning at low speed in a clinical centrifuge for 30-60 seconds. Aspirate off the buffer and spin at moderate speed in the clinical centrifuge for 30-60 seconds. Aspirate off residual buffer.

Crush eggs by spinning in the mini ultracentrifuge swinging bucket rotor (RP55S-344) at 10,000 rpm for 10 minutes at 2°C.

Remove the cytoplasmic fraction with a 1mL syringe and a 18-guage needle.

Add 1/1000 volume of LPC, 1/1000 volume of cytochalasin B or D, and 1/40 energy mix and 1/40 sucrose to the cytoplasmic fraction. Invert and flick gently to mix.

The extract is now ready for use.