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.

First, disrupt the disulfide bonds by incubating the solubilized protein in 25 microliters of TCEP, for 60 minutes at 55 degrees Celsius. Then, block free cysteines by incubating the solution with 12.5 microliters of the stock solution of NEM for three hours at room temperature.

Next, begin the process of chloroform-methanol precipitation. Transfer the protein solution from the 1.5-milliliter tube to a polypropylene or glass tube that can be centrifuged in a swinging bucket rotor at modest speed. Add 2 milliliters of methanol, and vortex briefly. Add 1 milliliter of chloroform, and vortex briefly. Then, add 1.5 milliliters of deionized water and vortex briefly again.

If necessary, invert the tube to ensure thorough mixing. Centrifuge the samples at 3,000 times G and 25 degrees Celsius for 30 minutes in a swinging bucket rotor. Carefully remove and discard the upper phase of the solution in a tube. Add 1.5 milliliters of methanol. Mix gently but thoroughly by gentle inversion of the tube, being careful not to fragment the opaque protein pancake.

Centrifuge the sample at 3,000 times G and 25 degrees Celsius for 10 minutes in a swinging bucket rotor. Using a glass serological pipette, remove as much of the top phase of the solution as possible without disturbing the protein pancake. Carefully rinse the pellet with 1 milliliter of methanol, and allow it to air dry for at least 10 minutes.

With the chloroform-methanol precipitation complete, begin the cleavage of the palmitoyl thioester linkages by resuspending the protein precipitate in 125 microliters of buffer A. Transfer to a new 1.5-milliliter tube. Sonicate the tube briefly, and then centrifuged it at 13,000 times G or higher and 25 degrees Celsius for 10 minutes to remove insoluble material.

Transfer the solubilized protein to new 1.5-milliliter tubes, and add 375 microliters of buffer H or buffer T. After incubating the samples for 60 minutes at room temperature, repeat the chloroform-methanol precipitation. To add mPEG to unprotected cysteines, begin by resuspending the pellet in 100 microliters of buffer A containing 10 millimolar TCEP.

Then transfer the suspension to a 1.5 milliliter tube. Add 25 microliters of stock mPEG 5k solution and mix by pipetting. Incubate at room temperature with end over end rotation. After 60 minutes of incubation, remove unincorporated mPEG 5k by performing chloroform methanol precipitation again.

Centrifuge at greater than 13,000 times G and 25 degrees Celsius for 10 minutes. Carefully remove the upper phase as before, avoiding the thick flocculent pancake. Add 1 milliliter of methanol and mix gently but thoroughly. Centrifuge at greater than 13,000 times G and 25 degrees Celsius for 10 minutes.

Carefully remove the supernatant and rinse the pellet with 1 milliliter of methanol. Again, centrifuge at greater than 13,000 times G and 25 degrees Celsius for 10 minutes. After air drying the pellet, resuspended in 50 microliters of buffer A without TCEP or any reducing agent.