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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of mouse brain membranes
- Decapitate mouse using a guillotine apparatus and dissect brain out rapidly (in <1 min, if possible, to minimize palmitoylation changes that may occur during dissection procedure). Homogenize immediately in 10 mL of homogenization buffer (Table 1) in a glass homogenizer (~12 strokes) on ice.
- Centrifuge lysates for 15 min at 1,400 x g at 4 °C. Transfer the supernatant to a new tube on ice. Resuspend the pellet (~6 strokes) in an equal volume of homogenization buffer and centrifuge at 710 x g for 10 min at 4 °C.
- Combine the supernatants from step 1.2 and centrifuge at 40,000 x g for 20 min at 4 °C.
- Discard the supernatant (cytosolic fraction) and resuspend the pelleted membrane (P2) fraction in homogenization buffer.
NOTE: Samples can be flash frozen and stored at -80 °C for later use.
- Perform a bicinchoninic acid (BCA) assay to quantitate protein levels. For the Acyl-PEGyl exchange gel-shift (APEGS) assay, begin with ~100−200 mg protein (maximum 250 mg) in a volume of 470 µL of buffer A (Table 1) in a 1.5 mL tube. Sonicate and centrifuge at >13,000 x g for 10 min at 25 °C to remove insoluble protein. Transfer solubilized protein to a new 1.5 mL tube.
2. Acyl-PEGyl exchange gel-shift (APEGS) assay
- Disrupt disulfide bonds and block free cysteines.
- Disrupt disulfide bonds by incubating in 25 mM tris(2-carboxyethyl)phosphine (TCEP; 25 µL, added from a stock solution; Table 1) for 60 min at 55 °C.
- Block free cysteines with 50 mM N-ethylmaleimide (NEM; 12.5 µL, added from a stock solution; Table 1) at room temperature for 3 h.
NOTE: The reaction can be extended overnight.
- Perform chloroform methanol (CM) precipitation.
- Transfer the protein solution from 1.5 mL tube to a polypropylene or glass tube that can be centrifuged in a swinging bucket rotor at modest speed. A 5 mL tube is ideal. Add four volumes (2 mL) of methanol (MeOH) and vortex briefly.
- Add two volumes (1 mL) of chloroform and vortex briefly.
- Add three volumes (1.5 mL) of deionized water (dH2O) and vortex briefly.
- Centrifuge the samples at 3,000 x g for 30 min at 25 °C in a swinging bucket rotor.
- Carefully remove and discard the upper phase.
- Add 3 volumes (1.5 mL) of MeOH and mix gently but thoroughly, being careful not to fragment the opaque protein pancake.
- Centrifuge at 3,000 x g for 10 min at 25 °C in a swinging bucket rotor.
- Using a glass serological pipet, remove as much of the top phase as possible without disturbing the protein pancake.
- Carefully rinse the pellet with 1 mL of MeOH.
- Air dry the pellet for at least 10 min.
NOTE: Dried pellet can be stored at -20 °C.
- Perform cleavage of palmitoyl-thioester linkages with hydroxylamine (NH2OH, HAM).
- Resuspend the protein precipitate in 125 µL of buffer A and sonicate briefly. Centrifuge at >13,000 x g for 10 min at 25 °C to remove insoluble material. Transfer solubilized protein to a new 1.5 mL tube.
- Add 375 µL of buffer H (HAM+; Table 1) or buffer T (HAM-; Table 1) and incubate samples for 60 min at 25 °C.
- Repeat the CM precipitation described in step 2.2.
NOTE: Dried pellet can be stored at -20 °C.
- Add mPEG to unprotected cysteines.
- Resuspend pellet in 100 µL of buffer A containing 10 mM TCEP. Transfer to a 1.5 mL tube.
NOTE: It is normal for significant loss of protein to have occurred during CM precipitation steps. All subsequent steps will be performed in a 1.5 mL tube, constraining the volume of protein to a maximum of ~120−130 µL. This will reduce protein loss during the final CM precipitation.
- Add 20 mM mPEG-5k (25 µL, added from a stock solution; Table 1) to protein sample and mix by pipetting. Incubate for 60 min at 25 °C with end-over-end rotation.
- To remove unincorporated mPEG-5k, perform CM precipitation as described in step 2.2 using these adjusted volumes: 4 volumes of MeOH (500 µL), 2 volumes of chloroform (250 µL), and 3 volumes of dH2O (375 µL). Centrifuge at >13,000 x g for 10 min at 25 °C.
- Carefully remove the upper phase as before, avoiding the thick, flocculent pancake. Add 1 mL of MeOH and mix gently but thoroughly. Centrifuge at >13,000 x g for 10 min at 25 °C.
- Carefully remove the supernatant and rinse the pellet with 1 mL of MeOH. Centrifuge at >13,000 x g for 10 min at 25 °C. Air dry pellet.
NOTE: Dried pellet can be stored at -20 °C.
- Resuspend the dried pellet in 50 µL of buffer A (without TCEP or any reducing agent).