Detecting Histone Modifications in Yeast Cells with Neurodegenerative Protein Overexpression

Begin with a suspension of yeast cells overexpressing proteins linked to neurodegenerative diseases.

This overexpressed protein is responsible for modifications in the DNA-packaging proteins or histones with chemical moieties.

Add sodium hydroxide and beta-mercaptoethanol and then incubate on ice. 

Sodium hydroxide and beta-mercaptoethanol lyse the cells, releasing the intracellular proteins, including histones.

Centrifuge the lysate to collect the proteins. Discard the supernatant.

Add a loading dye to the protein pellet and heat it to denature them.

Load these denatured proteins and a protein marker on the gel and electrophorese to separate them by size.

Electrotransfer the proteins onto a membrane, wash, and add a blocking buffer to prevent non-specific binding.

Add primary antibodies that specifically bind to modified histones.

Wash to remove unbound antibodies and incubate with fluorescent secondary antibodies, which bind to the primary antibodies.

A distinct fluorescent band confirms histone modifications, indicating overexpression of neurodegenerative proteins.

To lyse the cells for western blot analysis, thaw the yeast cell pellets on ice before resuspension in 100 microliters of distilled water. Add 300 microliters of 0.2 molar sodium hydroxide, and 20 microliters of 2-Mercaptoethanol to each cell sample. And resuspend the pellets by pipetting.

After a 10-minute incubation on ice, pellet the cells by centrifugation, and resuspend the samples in 100 microliters of loading dye. Then boil the samples for 10 minutes on a heating block. While the samples are being lysed, place two gels in a gel holder, and fill the inner chamber to the top with running buffer and the outside chamber to the two-gel line mark.

When the samples are ready, load 15 microliters each cell lysis solution into each well of the 10-well 12% polyacrylamide gel and add 5 microliters of protein ladder to the protein ladder lane well. Run the gel for approximately 45 minutes at 150 volts or until loading dye front reaches the bottom of the gel.

While the gel is running, soak two fiber pads per gel in transfer buffer, and one polyvinylidene fluoride or PVDF membrane per gel in methanol. When the gel is ready, rinse membrane and transfer buffer. And place one pre-soaked fiber pad on the bottom of a semi-dry transfer apparatus cell, followed by the PVDF membrane, the gel, and the second pre-soaked fiber pad.

Then set the power to 150 milliamps for 1 hour. At the end of the protein transfer, remove the membrane from the transfer apparatus for a brief rinse with distilled water. Place the rinsed membrane protein side up in a small staining box, and cover the membrane with Tris-buffered saline or TBS blocking buffer for a 1 hour incubation at room temperature with gentle rocking.

At the end of the blocking incubation, incubate the membrane overnight with an anti-yeast histone and modification specific antibody in TBS at 4 degrees Celsius, and a proper nuclear loading control antibody.

The next morning, wash the membrane four times in TBS, supplemented with 0.1% polysorbate 20 for five minutes with rocking at room temperature per wash. After the last wash, incubate the blot with the appropriate secondary antibodies for 1 hour at room temperature, followed by four 5-minute washes in TBST and one five minute wash in TBS alone with rocking.

Then image the blot on a fluorescent western blot imaging system for 2 minutes.