Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

One batch of ubiquitin remnant motif antibodies conjugated to protein A agarose bead slurry is split into six equal fractions. Dissolve the three peptide fractions according to the manuscript directions and spin down the debris.

Add the supernatants of the three fractions to the bead slurry while keeping the other three fractions on ice and incubate them for 2 hours at 4 degrees Celsius on a rotator unit.

Then, spin down the beads. Transfer the supernatant to a fresh batch of beads and repeat the incubation with the remaining three bead slurry fractions.

Store the supernatants for subsequent global proteome analysis and transfer the beads to 200-microliter pipette tips equipped with a GF/F filter plug.

Put the tips into 1.5-milliliter tubes equipped with a centrifuge tip adapter and wash the beads three times with 200 microlitres of ice-cold IAP buffer, followed by three washes with ice-cold purified water.

Spin down the columns at 200 x g for 2 minutes between washes, making sure not to let the column run dry. After the final wash, elute the peptides with two cycles of 50 microlitres of 0.15% TFA. Desalt the peptides with a C18 stage tip and dry them with vacuum centrifugation.