Assay of Apoptosis Using Fractionated Interphase Egg Extracts

3.4.1. Reconstitution Extract: Cytosolic and Heavy Membrane Fractions

1. Add ATP-regenerating mix (1/10 dilution of 0.2 Mphosphocreatine and 1/100 of 0.5 mg/mL creatine kinase and 0.2 M ATP ) to 170 |L of cytosolic extract.

2. Add 14 |L freshly prepared heavy membrane fraction enriched in mitochondria (Subheading 3.3.2.).

3. Incubate at room temperature. Withdraw samples at 30-min intervals to assess cytochrome c release and caspase activity (Subheadings 3.2.2.-3.2.3.; see Note 10).

3.4.2. Assessment of Cytochrome c-Induced Apoptosis

1. Thaw 50 to 100 |L cytosolic egg extract and add ATP-regenerating mix (1/10 dilution of 0.2 M phosphocreatine, 1/100 dilution of 0.5 mg/mL creatine kinase, and 1/100 dilution of 0.2 M ATP).

2. Prepare 10X working solutions of soluble cytochrome c to be added to the extract. Final concentrations of cytochrome c in the extract should range between 0.6 and 2 |g/mL (see Note 11).

3. Incubate at room temperature for 30 to 180 min.

4. Assess caspase activity as described in Subheading 3.2.3.

3.5. Modification of the Egg Extract Through Immunodepletion of Proteins, Addition of Recombinant Proteins, or Neutralizing Antibodies

The Xenopus egg extract in vitro reconstitution systems provide the opportunity to easily study the function of proteins through their removal, addition, or inhibition. Complete immunodepletion of proteins from the egg extract often requires multiple rounds of depletion using a high-affinity antibody. To ensure that the process of immunodepletion has had no effect on the process of apoptosis in the extract, it is critical to use preimmune or IgG-depleted extract as a control. It is also imperative that the extract not be diluted during the depletion process so the function of the extract is not compromised.

Supplementation of the extract with wild-type or mutant recombinant proteins offers an easy assessment of their apoptotic function. For example, preincubation of a cytosolic extract with recombinant MAPK, a known inhibitor of apoptosis, inhibits cytochrome c-induced caspase activity (6); addition of Reaper to a crude interphase extract induces rapid full-blown apoptosis (2).

Although depletion of proteins from the egg extract can provide a very useful means to assess the function of a particular protein factor, sometimes it is not possible to remove sufficient protein from the egg extract. In this case, addition of an antibody specific for the protein of interest may act to neutralize the function of the protein. For example, addition of an antibody to Weel, a protein required for the spontaneous apoptosis program in the egg extract, completely inhibited caspase activity in the crude interphase extract (8). The protocols described next outline how to deplete or supplement the egg extract without compromising the ability of the extract to function properly.

3.5.1. Immunodepletion From the Egg Extract

1. Precouple the antibody to the beads by incubating the antibody with protein A (or protein G) Sepharose in PBS containing 2 mg/mL BSA fraction V. (Incubation times and temperature vary depending on the antibody used.) Prepare control beads (preimmune or IgG) in the same manner. Although approximate volumes may vary depending on the antisera used, approx 15 ||L packed beads can be incubated with approx 10 |lg of IgG in 200 |L PBS containing 2 mg/mL BSA.

2. Pellet the beads using an ultracentrifuge; wash twice with ELB. Remove all of the buffer from the beads using a 25-gage needle attached to a 1-mL syringe.

3. Add 100 |L extract to be depleted to the packed antibody beads and rotate at 4°C for 40 min.

4. Pellet the beads by centrifugation and remove the supernatant to a new tube of prewashed antibody beads.

5. Rotate for an additional 40 min at 4°C

6. Rinse a Bio-Spin column with ELB. Be very careful to dry the column after rinsing to prevent dilution of the extract.

7. Transfer the extract/beads to the Bio-Spin column and spin for 3 min at high speed in a clinical centrifuge. This step ensures the removal of the beads from the extract.

8. The depleted extract is now ready to be used to assess apoptosis as described in Subheadings 3.2. and 3.4. At this point, the extract has been depleted twice. Depending on the protein of interest and the antibody, the number of depletions and length of incubation of extract with antibody beads will vary. You will have to optimize the depletion protocol for each protein of interest. To assess how well the depletion worked, perform an immunoblot for the protein of interest.

3.5.2. Addition of Recombinant Protein and Neutralizing Antibodies to Egg Extracts Addition of Recombinant Proteins

1. It is best if the recombinant protein is dialyzed into XB buffer. To maintain the integrity of the egg extract, add the protein at a 1/5 dilution or greater into the egg extract (1/10 or more is best).

2. Incubate the protein in the extract and assess apoptosis as described in previous subheadings. Addition of Neutralizing Antibody

1. To inhibit the protein of interest with antibody, add 0.5 to 1 mg/mL antibody into the egg extract. Rotate the extract/antibody for 20 min at 4°C (see Note 12).

2. Add ATP-regenerating mix and assess apoptosis as described in previous subheadings.

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