A phosphocellulose flowthrough fraction required for accurate transcription in vitro by RNA polymerase II was found to contain a DNase inhibitor which was necessary to maintain template integrity (Price D.H., Sluder A.E. & Greenleaf A.L. (1987) J. Biol. Chem. 262, 3244-3255). Starting with a Drosophila Kc cell nuclear extract, the DNase inhibitory activity has been purified 19,000-fold. In combination with the other necessary fractions, the highly purified inhibitor continues to support reconstruction of transcription. It thus appears to be the only required activity in the original phosphocellulose flowthrough fraction. The inhibitor is a protein which does not bind to DNA or inhibit DNase I, so that it has also been useful in assays for DNA binding proteins in crude, DNase-contaminated fractions.