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.