As cells persist in their environment, they are exposed to harmful agents that. can damage their genomic DNA. When DNA becomes damaged, p53, a tumor suppressor, is stabilized and acts as a transcription factor to cause either cell cycle arrest or apoptosis. Strict p53 regulatory mechanisms have been well characterized relative to phosphorylation and dephosphorylation, but acetylation of p53 in response to DNA damage has also been shown to participate in p53 function. Proper investigation of the many roles that acetylated p53 plays in the cell requires accurate in vitro studies, which can only be easily conducted if highly pure acetyl-p53 is available. Purified p53 that is acetylated in vitro can routinely achieve 10-20%. Separating this acetylated fraction from the undesired unacetylated fraction can be technically challenging, inefficient, and time consuming. We have developed an in vivo strategy to rapidly produce microgram quantities of p53 preparations that are greater than 60% acetylated using co-infection of p53 and p300 baculoviruses in Sf21 insect cell culture. Immunoaffinity recovery followed by further depletion of unacetylated p53 results in a preparation that is greater than 70-75% in acetyl-p53 after a single round, and undetectable levels of unacetylated p53 after two rounds. This approach to preparing acetylated protein in vivo may also extend to other acetylated transcription factors and histories. (c) 2004 Elsevier Inc. All rights reserved.