Assessing the specificity of PROTACs and confirming their proposed mechanism of action are critical for a robust targeted protein degradation program. Owing to their novel mechanism, new assays are needed to meet these goals. We and others have shown that a common explanation of PROTAC efficacy is the ability of the PROTAC to form a ternary complex between the E3 ubiquitin ligase and the target protein. In this chapter, we provide a simple in vitro method to quickly and inexpensively assess this property of PROTAC molecules. We provide detailed instructions for the purification of the specific E3 ubiquitin ligase VHL and then a generic protocol which can be adapted to any E3 ligase and substrate protein combination. This accessible method to study the ternary complex can strengthen any PROTAC-focused medicinal chemistry effort.
Keywords: Degrader; E3 ubiquitin ligase; PROTAC; Targeted protein degradation; Ternary complex; VHL.
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