Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue

Take cell membrane proteins from mouse brain tissue.

These proteins exhibit post-translational modification with palmitoyl groups, a fatty acid, attached to cysteine residues.

Add a reducing buffer and incubate at a high temperature to break bonds between non-palmitoylated cysteines, exposing them.

Incubate with a blocking reagent that binds to the exposed cysteines, leaving only the palmitoylated cysteines reactive.

Transfer the solution, mix it with chloroform, methanol, and deionized water, centrifuge it to precipitate the proteins, and remove any impurities. Next, add methanol, centrifuge, and discard any remaining contaminants.

Add a buffer, sonicate to solubilize the pellet, then centrifuge and remove insoluble materials.

Apply a cleaving reagent to remove the palmitoyl groups from the palmitoylated cysteines.

Repeat chloroform-methanol precipitation and remove any impurities.

Incubate with a polymer that binds to unblocked cysteines, helping to identify the palmitoylated proteins.

Repeat chloroform-methanol precipitation and remove any unbound polymers.

Resuspend the pellet for further analysis of the palmitoylation state of the isolated proteins.