An affinity purification procedure to isolate oxidized p53.

ABSTRACT

Oxidation of cysteine is now known to serve as a fundamental mechanism to control protein function or activity. Many redox-regulated proteins do not oxidize to homogeneity, resulting in a mixture of reduced and oxidized species which cannot be separated chromatographically. Here we describe a protocol for the separation of reduced and oxidized forms of the tumor suppressor protein p53. This purification method relies on the reversible labeling of thiol groups with biotin and exploitation of the ultrastrong biotin-avidin interaction. This purification procedure can be applied to other cysteine-containing proteins where enrichment of the oxidized form is required.