Affinity Purification of Ubiquitinated Proteins Using p62-Agarose to Assess Ubiquitination of Clade A PP2Cs

Certain E3 ubiquitin ligases play a key role in the abscisic acid (ABA) pathway by targeting clade A type 2C protein phosphatases (PP2Cs) for degradation. At early stages of ABA signaling, degradation of PP2Cs is a complementary step to PP2Cs inhibition by ABA receptors. At later steps, protein levels of PP2Cs are increased as a negative feedback mechanism. Subsequently, E3 ligases targeting PP2Cs are critical to recover the basal PP2C levels and reset the ABA signaling. BTB/POZ AND MATH DOMAIN proteins (BPMs) are substrate adaptors of a multimeric cullin3-RING based E3 ligase and target for degradation clade A PP2Cs. In this chapter, we provide a detailed protocol to assess the ubiquitination of PP2CA, a clade A PP2C, mediated by BPMs using agarose-immobilised p62-derived ubiquitin-associated (UBA) domain, which efficiently binds ubiquitinated proteins.