Timing: 1 day

In this step, biotin tag is added to the newly available free thiol sites which were previously palmitoylated for affinity enrichment. The palmitoylated proteins can be detected using antibody against the protein of interest or if the protein was co-expressed with a tag on it.

Dissolve the protein pellet in 200 μL of flex buffer supplemented with 20 mM Biotin-HPDP.

Incubate the samples at 22°C–24°C with end-over-end rotation for 1 h.

Perform protein precipitation one final time as described in steps 19–24.

Dissolve the protein in 100 μL of flex buffer.

Estimate protein concentration by mini-BCA assay (Figure 1C) using a 10 μL protein sample.

Normalize protein concentration for any variability across samples.

Remove a 20 μL aliquot from each sample to load as an input control. Mix the sample with 20 μL of 2x SDS-PAGE Laemmli buffer and store at −20°C until ready to load.

Dilute the remaining 70 μL sample 10 times by adding 630 μL of core buffer. This dilutes the SDS concentration to 0.1%.

Add 30 μL of pre-washed streptavidin agarose beads directly to each sample and vortex for ∼10 s to mix the contents.

To first wash and prepare the streptavidin-agarose beads, add 30 μL agarose beads per sample to 500 μL of core buffer.

Spin at 10,000 g for 2 min at room temperature.

Wash the agarose beads for a total of 3 times taking 500 μL core buffer each time. After the final wash step, resuspend the beads in 30 μL of core buffer per sample.

Incubate the samples with end-over-end rotation at 4°C for 18 h–24 h.

The following day, wash the samples with 500 μL of core buffer supplemented with 0.1% SDS and 0.1% NP-40.

Repeat washes for a total of 3 times, spinning the samples at 12,000 g for 5 min between each of the washes.

To the pellet (Figure 1D), add 75 μL of 1x Laemmli buffer and store at −20°C until ready for SDS-PAGE.

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