Preparation and Fractionation of Xenopus Laevis Egg Extracts

Create a transfer pipette by breaking a Pasteur pipette and inserting it into a pipette bulb. Transfer the dejellied eggs into a 15-ml polypropylene tube using the wide-mouth pipette to avoid lysing them.

After centrifuging eggs briefly and removing buffer, add aprotinin, leupeptin, cytochalasin B (5 μg/ml), and cycloheximide (50 μg/ml) directly onto the packed eggs to inhibit proteases, actin polymerization, and protein synthesis.

Centrifuge the eggs for 15 minutes at 12,000 g in a Sorvall HB4 swinging bucket rotor at 4°C. This separates the lysate into three layers: yellow lipid on top, crude extract in the middle, and dark pellet at the bottom. Carefully remove the crude extract using an 18-gauge needle and 3-ml syringe.

Transfer crude extract to a 2.5-ml ultraclear tube and centrifuge for 90 minutes at 55,000 rpm (250,000 g) at 4°C in a TLS 55 rotor. This separates the extract into a clear soluble portion, pale yellow light membrane fraction, dark membrane fraction with organelles, and a ribosome/glycogen pellet.

Carefully remove the clear soluble fraction with a cut-off 200-microliter pipette tip, keeping the extract on ice. Transfer to a new 2.5-ml ultraclear tube and centrifuge for 25 minutes at 250,000 g at 4°C to further purify the cytoplasmic fraction.

Collect the top clear soluble fraction and divide it into 100-microliter aliquots. Freeze the aliquots in liquid nitrogen and store at -80°C until ready for use.

Carefully remove the light membrane fraction from the first ultracentrifugation using a cut-off pipette tip. Transfer to a 2.5-ml tube containing 1.5 ml egg lysis buffer and gently disperse membranes by pipetting and inverting with parafilm.

Underlay the membranes with 2x sucrose egg lysis buffer using a Pasteur pipette to create a cushion that removes peripherally-associated proteins. Centrifuge for 20 minutes at 25,000 g at 4°C, then remove the supernatant, collect the membrane pellet, and freeze aliquots in liquid nitrogen for storage at -80°C.