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 dis