Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative

Treat mouse cortical astrocytes with a cleavable biotin derivative.

Incubate at a low temperature to prevent protein internalization and facilitate biotin attachment to the target surface proteins.

Wash and add a warm medium, then incubate to induce internalization of the biotinylated proteins.

Wash and treat with a membrane-impermeable reducing agent to cleave the non-internalized biotin.

Add a quenching reagent to stop the reaction and wash.

Harvest the cells and centrifuge. Remove the supernatant.

Add a lysis buffer to lyse the cells, releasing the non-biotinylated and internalized biotinylated proteins.

Centrifuge to pellet the cell debris.

Collect the supernatant containing proteins, add streptavidin-agarose beads, and mix to capture the biotinylated proteins.

Centrifuge to pellet the beads and discard the supernatant.

Add a loading buffer and heat to denature and release the proteins from the beads.

Run the protein on a gel and transfer it onto a blotting membrane.

Detect using primary and secondary antibodies to visualize a distinct protein band, confirming successful protein internalization.