This biochemical method allows for the determination of the palmitoylation state of any membrane protein expressed in the brain for which a suitable antibody is available. The main advantage of this technique is that it does not require affinity purification and instead utilizes changes in gel mobility to determine the number of modifications of a protein of interest. After dissecting out the brain, homogenize it immediately in 10 milliliters of homogeneization buffer in a glass homogenizer on ice.
Using approximately 12 strokes, centrifuge the lysates for 15 minutes at 1400 times G, and four degrees Celsius. Transfer the supernatant to a new tube on ice. Resuspend the pellet in 10 milliliters of homogeneization buffer.
Homogenize it using approximately six strokes. Centrifuge the suspension at 710 times G and four degrees Celsius for 10 minutes. Combine the supernatants and centrifuge them at 40, 000 times G and four degrees Celsius for 20 minutes.
Discard the supernatant and resuspend the pelleted membrane fraction in a homogeneization buffer by breaking up the pellet with a pipette tip and pipetting up and down. Perform ABCA assay to quantitate protein levels. To perform the APEGS assay, place 470 microliters of buffer A in a 1.5 milliliter tube and add approximately 100 to 200 milligrams of protein.
Sonicate the tube and then separate the insoluble protein by centrifuging the tube at 25 degrees Celsius and a minimum of 13, 000 times G for 10 minutes. Transfer the solubalized protein to a new 1.5 milliliter tube. First, disrupt the disulphide bonds by incubating the solubalized 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 two milliliters of methanol and vortex briefly. Add one 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 3000 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 3000 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 one 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 centrifuge it at 13, 000 times G or higher and 25 degrees Celsius for 10 minutes to remove insoluble material. Transfer the solubalized 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 m-PEG to unprotected cysteines, begin by resuspending the pellet in 100 microliters of buffer A containing 10 mil or more TCEP.
Then transfer the suspension to a 1.5 milliliter tube. Add 25 microliters of stock m-PEG 5K solution and mix by pipetting. Incubate at room temperature with end over end rotation.
After 60 minutes of incubation, remove unincorporated m-PEG 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 one 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 one milliliter of methanol.
Again, centrifuge at greater than 13, 000 times G and 25 degrees Celsius for 10 minutes. After air drying the pellet, resuspend it in 50 microliters of buffer A without TCEP or any reducing agent. Reserve 5 microliters of the solution for BCA protein quantitation.
After quantitation, add an appropriate amount of 4x Laemmli sample buffer. Load the sample onto an SDS page gel. Use standard separation transfer and detection protocols for western blotting.
To investigate the palmitoylation state of SynDIG proteins, P2 membrane fractions from homogenized mouse brains were subjected to the APEGS assay. Separation and probing with antibodies demonstrated that SynDIG1, SynDIG4 Prrt1, and Prrt2 are palmitoylated in the mouse brain. The purpose of the chloroform methanol precipitation is to prevent one chemical, such as NEM, interfering with the next step of the protocol.
A proper chloroform methanol precipitation will remove undesirable chemicals while minimizing protein loss. This method can be applied to brain lysates from mice of different ages or from different brain regions to investigate the spatial-temporal regulations of palmitoylation.
