Separation of transcriptively active and inactive chromatin Agarose gel chromatography

Abstract

Sheared chromatin fractionated by currently accepted methods of agarose gel exclusion chromatography, undergoes a limited and non-specific aggregation resulting from the high ionic strength and divalent cation concentration of the column elution buffer. Such aggregation causes the artifactual appearance of radioactively labeled, newly synthesized RNA within the column exclusion volume, erroneously suggesting an enrichment for actively transcribed chromatin. Claims for the efficacy of agarose gel exclusion as a method for separating template-active and -inactive chromatin are based largely on assays for active chromatin which rely on localization of specific molecular complexes of chromatin and nascent RNA. Under the conditions employed, the present studies invalidate this assay and thus cast considerable doubt on the agarose gel exclusion method itself.