p53 levels and activity are controlled in large part through regulated 
ubiquitination and subsequent destruction by the 26S proteasome. 
Monoubiquitination of p53 is mediated primarily by the RING-finger E3 ubiquitin 
ligase MDM2 and impacts p53 activity through modulation of p53 localization and 
transcription activities. Recently, several E4 ubiquitin ligases (E4s) have been 
identified which serve to extend these monoubiquitin chains. The ubiquitin 
ligase activity of these factors toward p53, and their contribution to p53 
degradation, can be studied using a variety of in vitro and in vivo methods and 
reagents which will be described in this chapter. These methods include in vivo 
ubiquitination of p53 using HA-ubiquitin or his-ubiquitin; the in vitro E3 
ubiquitin ligase assay, in which ubiquitin reaction components (URC) are 
incubated with a purified E3 or E4 ligase; a one-step E4 assay, in which URC are 
incubated with a substrate, E3, and E4; and a two-step E4 assay in which p53 is 
monoubiquitinated in an E3 reaction, and subsequently purified and incubated 
with an E4. Finally, we will describe an in vitro degradation assay in which 
ubiquitinated p53 is incubated with purified 26S proteasomes. Together, these 
assays can be used to provide insight into the biochemical nature of p53 
ubiquitination and degradation.