Protein S-acylation is a post-translational modification in which a fatty acid, usually palmitic acid, is thioesterified to the cysteine thiol. Palmitoylation, a type of S-acylation, is reversible lipid modification that allows regulated membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking.

The RayBio® S-Acylation Detection Kit uses a modified 'biotin-switch' (acyl-biotinyl exchange (ABE)) method to allow for the direct visualization of acylated (palmitoylated) proteins by western blot analysis. In this S-acylation biotin switch protocol, unmodified free cysteines are first blocked. Acylated cysteines are then selectively reduced for specific labeling with biotin-maleimide reagents, which irreversibly bind to the cysteine thiol that was acylated. Biotinylation of the newly formed thiol groups can then be detected by western blot or other immunoassay. In addition, avidin resin can be used to selectively enrich acylated proteins/peptides labeled with biotin. This procedure also allows for S-acylation or S-palmitoylation site mapping by using mass spectrometry.

1. Acetone, ≥ 98% (hazardous)
2. 1.5 ml microcentrifuge tubes
3. 15 ml tubes (polypropylene)
4. 10 ml graduated cylinders (X2)
5. Benchtop centrifuge and microcentrifuge (4°C)
6. Water bath or heat block
7. Distilled or deionized water
8. Precision pipettes to deliver 2 µl to 1 ml volumes
9. Adjustable 1-25 ml pipettes for reagent preparation

1. Prepare all reagents and samples as instructed.
2. Block unmodified free cysteines using prepared S-Acylation Blocking Buffer at 50°C with gentle rocking.
3. Precipitate proteins with ice-cold acetone.
4. Wash protein pellet twice with cold acetone/water mixture.
5. Resuspend protein pellets in Buffer B containing Reducing and Labeling Reagents.
6. Precipitate proteins with ice-cold acetone.
7. Resuspend protein pellets.