Detection of Phosphorylated Tau Proteins in Mouse Brain Samples Using Western Blot Analysis

Start with a mouse brain tissue in an extraction buffer containing protease inhibitors and a detergent.

Homogenize the tissue to release proteins into the buffer. The protease inhibitors block protease activity, preventing protein degradation.

Centrifuge and collect the supernatant with proteins.

Add a loading buffer containing detergent and a reducing agent to denature the protein.

Load the proteins from the control and sample onto an SDS-PAGE gel. Apply an electric field to separate proteins into distinct bands.

After electrophoresis, transfer protein bands onto a membrane.

Treat the membrane with a buffer containing fat-free milk to block non-specific interaction sites.

Incubate with primary antibodies that bind specifically to phosphorylated tau proteins. 

Wash and Incubate with enzyme-conjugated secondary antibodies.

Add a substrate that reacts with the enzyme to produce a light signal, confirming the phosphorylated tau proteins.

An intense luminous band in the sample compared to the control suggests neurodegenerative pathology.