Figure 4

Highly purified HatB3.1 contains Gcn5p and Ada3p in a high molecular weight complex. A) Flowchart outlining the partial purification of HatB3.1. B) Fluorograms of liquid HAT assays of the Superose 6 column fractionation of HatB3.1 activity (top 2 panels). Assays used either free histones or chromatin as substrate (as indicated). The relative elution of molecular weight standards is shown along the top, while the migration of histones H3 and H4 is indicated at the right. Column fraction aliquots (15 μL) were also resolved by SDS-PAGE and visualized by silver staining (bottom panel, protein ladder mobility is represented at right). Corresponding fraction numbers for both the fluorograms and silver stained gel are indicated along the bottom. C) Peak HatB3.1 containing Superose 6 fractions, as indicated at top of blots, were resolved by three identical 10% SDS gels, transferred to nitrocellulose and probed with the indicated antibodies (left of blots). Presence of Gcn5p, Ada2p and Ada3p in nuclear extract and/or column fractions was visualized via chemifluoresence. Relative migration of protein standards is shown on the right.