In a previous video, we demonstrated how to obtain and de jelly eggs from mature XUS labis. Females crude extracts from these eggs provide a useful source of cytoplasmic and organelle material for the in vitro reconstitution of cellular events such as nuclear assembly, nuclear protein import, and DNA replication. Hi, my name's Marie Cross and I'm a graduate student in Dr.Maureen Power's lab in the Department of Cell Biology at Emory University in Atlanta, Georgia.
Today we will show you how to fractionate egg extract into soluble cytoplasmic and light membrane fractions. This procedure involves the following steps, lysing the eggs, collecting the crude extracts, fractionating crude egg extracts, and collecting cytoplasmic extract and collecting the light membrane fraction of the egg extract. So let's get started.
Let's begin by lysing eggs and collecting crude extracts before egg lysis. A transfer pipette must be made by breaking a pasture pipette and then carefully inserting the broken end into a pipette bulb. Transfer the eggs into a 15 mil polypropylene tube.
A wide mouth pipette is required to avoid lysing the eggs. Next, centrifuge the eggs for 30 seconds. In a clinical centrifuge at 400 times G, remove egg buffer from the top of the packed eggs by aspiration.
Note the volume of the packed eggs. Based on this volume, add a solution of aprotinin and leptin and then cytokine B directly on top of the eggs and a final concentration of five micrograms per milliliter using five milligram per milliliter stock solutions also add cyclo heide at a final concentration of 50 micrograms per mil using a 10 milligram per milliliter stock solution. Aprotinin and leptin inhibit proteases.
Cytokine B inhibits actin polymerization and cyclo heide inhibits protein synthesis. Now lyce the eggs by centrifusion for 15 minutes at 12, 000 Gs in the sova HB four swinging bucket rotor at four degrees Celsius. The lys eggs will be separated into three layers.
A yellow lipid layer on top, the crude extract in the middle, and a dark pellet. Remove the crude extract by piercing the side of the polypropylene tube with an 18 gauge needle attached to a three mill syringe. The crude extract contains nuclear and cytosolic components, light membrane from the er, nuclear envelope, ribosomes, and heavy membrane organelles, including mitochondria.
The extract can be stored for one to two hours on ice. I've just shown you how to make crude extract from opus lavis eggs. It's important to remember that these extracts will not support nuclear assembly once they've been frozen and thawed unless they've been fractionated into the cytoplasmic and light membrane fractions.
I'll show you how to do that now. To begin fractionation, transfer the crude egg extract to a 2.5 milliliter UltraClear centrifuge tube. Centrifuge the egg extract for 90 minutes in a Beckman TLS 100 centrifuge using a TLS 55 rotor at 55, 000 RPMs or 250, 000 Gs at four degrees Celsius.
After centrifugation, the extract will separate into a clear soluble portion with a pale yellow membrane fraction. Below a dark membrane fraction consisting of mitochondria and other organelles will lay below the light membranes. At the bottom is a large fraction containing ribosomes and glycogen, keeping the extract on ice.
Remove the clear soluble fraction with a cutoff 200 microliter pipette tip. Take care not to disrupt the light membrane layer, retain both the cytoplasmic fraction in the membrane layer and keep on ice. Next, centrifuge the soluble fraction again in a new 2.5 mil UltraClear centrifuge tube for 25 minutes at 250, 000 Gs at four degrees Celsius after centrifugation, again, collect a top soluble fraction, then divide it into a hundred microliter aliquots, freeze the aliquots and liquid nitrogen and store at minus 80 degrees Celsius until ready for use.
Now I'll demonstrate how to collect the light membrane fraction from the opus egg extract Using a micro pipetter with a cutoff or wide bore pipette tip. Carefully remove the light membrane fraction from the membrane layer leaving dark membranes undisturbed. Transfer the light membranes to a 2.5 UltraClear tube containing 1.5 mils egg lysis buffer.
Begin to disperse the membranes and buffer by pipetting three times with a one mil micro pipetter tip. Then hold perfil over the top of the tube and invert several times to gently complete dispersion of membranes into buffer. Then underlay the membranes with two x sucrose egg lysis buffer by releasing it from a pasture pipette at the bottom of the membrane containing tube.
Add enough buffer to fill the tube. A clear layer will easily be seen below the membranes. The two x sucrose egg lysis buffer acts as a cushion to remove any proteins peripherally or non-specifically associated with the membranes centrifuge 20 minutes and 25, 000 Gs in the TLS 55 rotor at four degrees Celsius.
After centrifugation, the membrane will be in a loose yellow pellet at the bottom of the tube, pipette off the clear buffer until only the membrane fraction remains in the tube with a wide bore pipette tip. Collect a membrane and distributed into aliquots, then flash, freeze, and liquid nitrogen and store at minus 80 degrees Celsius. Now these fractions can be used for various purposes, including the formation of nuclei.
In vitro. We've just shown you how to prepare crude extract from xenopus lavis eggs and how to fractionate them into cytoplasmic and light membrane fractions, either freshly prepared crude extract or frozen soluble plus light membrane fractions can be used for in vitro nuclear assembly. Please see our other video in which we demonstrate in vitro nuclear assembly in these extracts.
So that's it. Thanks for watching and good luck with your experiment.
