Direct assay for O6-methylguanine-acceptor protein in cell extracts

E. A. Waldstein*, E. H. Cao, R. B. Setlow

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

A direct in vitro assay for O6-methylguanine-acceptor protein in cell extracts that measures the transfer of radioactivity from labeled O6-methylguanine (O6MeGua) adducts in an exogenous DNA substrate to protein is described. The protein-bound radioactivity is released and separated from that remaining in the DNA by sequential digestion with protease K and aminopeptidase M, and appears in the alcohol-soluble fraction of the digest. Data obtained by the direct assay are similar to those obtained by an indirect assay that measures the amount of O6MeGua-acceptor protein as the loss of O6MeGua from the DNA. In addition to its accuracy, the direct assay is also simple and can measure the amount of O6MeGua-acceptor activity in cell extracts prepared from as few as 0.5-1.0 × 106 mammalian cells.

Original languageEnglish
Pages (from-to)268-272
Number of pages5
JournalAnalytical Biochemistry
Volume126
Issue number2
DOIs
StatePublished - 1 Nov 1982
Externally publishedYes

Funding

FundersFunder number
U.S. Department of Energy

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