Detection of Misfolded Prion Protein Aggregates in Mouse Brain Tissue Using Western Blotting

Take tissue homogenate from a mammalian brain that contains both normal prion proteins and misfolded prion aggregates responsible for neurodegenerative diseases.

Incubate with a proteolytic enzyme to digest the normal prions, leaving the resistant aggregates intact.

Treat the aggregates with a denaturing buffer at a high temperature to linearize the proteins and impart a uniform negative charge.

Load the sample onto an electrophoresis gel and separate the proteins into bands, which confirms the presence of aggregates.

Using an electric field, transfer the separated proteins onto a membrane.

Incubate the membrane in a blocking solution to mask non-specific binding sites.

Incubate with peroxidase enzyme-tagged antibodies, which bind specifically to the prion aggregates.

Wash the membrane to remove unbound antibodies, minimizing non-specific signals.

Add a chemiluminescent substrate and an oxidizing agent, enabling the peroxidase to oxidize the substrate and produce light.

Measure the light intensity to quantify misfolded prions in the homogenate.