Protocol for Detection of Palmitoylated Proteins

Protocol for Detection of Palmitoylated Proteins

1. Detection via Click Chemistry Reaction

1.1 Procedure

1.1.1

Seed cells in a 15-cm dish at 2 × 10⁶ cells per dish. For metabolic labeling, incubate the cells with Alk-16 (palmitic acid-15-yne) at a final concentration of 50 µM in pre-warmed DMEM supplemented with 2 % (v/v) charcoal-stripped BI FBS and 1 % penicillin-streptomycin. Use DMSO-treated cells as a control. To ensure proper dissolution of Alk-16, sonicate or vigorously vortex the medium before addition. Perform the labeling for 18-24 h.

1.1.2 

Harvest the Alk-16-labeled cells and wash them twice with ice-cold PBS by centrifugation at 550 × g, 4 °C.

1.1.3 

Resuspend the cell pellet in 500 µL of PBS containing 1 ×cOmplete™ EDTA-free protease inhibitor cocktail (Roche). Lyse the cells by sonication on ice at 30 % amplitude, using 2 s “on” / 1 s “off” pulses for a total of 2 min.

1.1.4 

Centrifuge the lysate at 18,000 × g for 1 h at 4 °C to collect the plasma membrane fraction.

1.1.5 

Resuspend the pellet from both Alk-16-labeled and DMSO-treated samples in 250 µL of PBS containing 1 % Triton X‑100 and 1× cOmplete™ EDTA-free protease inhibitor cocktail. Rotate the suspension at 4 °C for 1 h.

1.1.6 

Centrifuge the lysate at 13,000 rpm for 30 min at 4 °C. Transfer 225 µL of the supernatant to a clean microcentrifuge tube; retain 20–30 µL of the supernatant as the input sample.

1.1.7 

Add the CuAAC reaction components to the supernatant to bring the final volume to 250 µL (see Section 3.2 for reactant concentrations). Incubate the reaction mixture at room temperature with rotation for 1.5 h.

1.1.8 

While the reaction proceeds, wash 160 µL of streptavidin-agarose bead slurry (adjusted with storage buffer) three times with lysis buffer.

1.1.9 

After the CuAAC reaction, if any insoluble material is present, briefly centrifuge the mixture at 12,000 rpm for 30 s. Transfer the entire clarified reaction mixture to the pre‑washed beads and rotate at 4 °C for 1 h to allow binding.

1.1.10 

Collect the beads by centrifugation at <3,000 rpm, 4 °C. Wash the beads five times with 600 µL of lysis buffer, gradually reducing the Triton X‑100 concentration from 1 % to 0 % over successive washes, to remove non‑specifically bound probes and proteins.

1.1.11 

Add 80 µL of lysis buffer and 20 µL of 5× protein loading buffer to the beads to prepare pull‑down samples. For input samples, adjust the volume with lysis buffer and loading buffer to a final total of 50 µL.

1.1.12 

Load 10 µL of input and 20 µL of pull‑down samples onto a 12 % SDS‑PAGE gel and resolve by electrophoresis. Detect palmitoylated proteins by Western blotting.

2. Detection via Acyl‑Biotin Exchange (ABE) Reaction

2.1 Upstream Immunoprecipitation (IP)

2.1.1

Harvest cells and lyse them on ice for 30 min in 300–1000 µL of mild lysis buffer (50 mM Tris‑HCl, pH 7.5, 150 mM NaCl, 1 % Triton X‑100, 1× cOmplete™ protease inhibitor cocktail (Roche), and 1 mM PMSF). Clear the lysates by centrifugation at 14,000 × g for 15 min at 4 °C, and collect the supernatants for immunoprecipitation.

2.1.2 

Add the primary antibody to the supernatants and incubate for 2 h or overnight at 4 °C to form antigen‑antibody complexes.

2.1.3

Transfer 20 µL of Pierce™ Protein A/G Magnetic Beads to a 1.5‑mL microcentrifuge tube. Add 1 mL of mild lysis buffer, gently vortex to mix, place the tube on a magnetic stand to collect the beads, and discard the supernatant.

2.1.4 

Wash the beads twice with mild lysis buffer, then once with mild lysis buffer containing 1× protease inhibitor cocktail.

2.1.5

Add the antigen‑antibody mixture to the tube containing the pre‑washed magnetic beads. Incubate at room temperature for 2–4 h with gentle mixing.

2.1.6 

Collect the beads on the magnetic stand and remove the flow‑through (which may be saved for analysis if desired). Add 1 mL of mild lysis buffer, mix gently, collect the beads, and discard the supernatant. Repeat this wash step four times in total.

2.2 Downstream ABE Reaction (Using Kit)

2.2.1 

Remove the supernatant from step 2.1.6. Add 500 µL of Solution Ⅰ and mix by inverting the tube 15 times. Place on the magnetic stand, capture the beads, and discard the supernatant.

2.2.2 

Add 300 µL of Solution Ⅰ and 100 µL of Solution Ⅱ. Rotate at room temperature for 30 min. Capture the beads on the magnet and discard the supernatant.

2.2.3 

Add 485 µL of Solution Ⅰ, 10 µL of Solution Ⅳ, and protease inhibitor cocktail. Rotate at room temperature for 30 min or at 4 °C overnight. Capture the beads and discard the supernatant.

2.2.4 

Wash the beads five times with Solution Ⅰ, capturing the beads on the magnet and discarding the supernatant each time.

2.2.5 

Add 400 µL of Solution Ⅲ and evenly split the bead suspension into two tubes. Label the tubes "–HAM" and "+HAM".

2.2.6 

Add 200 µL of Solution Ⅴ to the +HAM tube, and 200 µL of Solution Ⅵ to the –HAM tube. Rotate at room temperature in the dark for 1 h. Capture the beads on the magnet and discard the supernatant.

2.2.7 

Add 480 µL of Solution Ⅲ and 20 µL of Solution Ⅶ to each tube. Rotate at room temperature in the dark for 1 h. Capture the beads and discard the supernatant.

2.2.8 

Wash the beads three times with Solution Ⅲ, collecting the beads on the magnet and discarding the supernatant after each wash.

2.2.9

Add 100 µL of 1× SDS‑loading buffer to each tube and heat the samples at 95 °C for 10–15 min in a heating block. Magnetically separate the beads and transfer the supernatant (containing the target antigen) to a new tube. Assess the palmitoylation level of the protein by Western blotting using the primary antibody (Solution Ⅷ).

3. Materials

3.1 Reagent Information

3.2 CuAAC Reaction Mix (per 250‑µL reaction)