Uno q column

TFIIK and holo-TFIIH were purified from yeast as previously described (23 (link)) with minor modifications. In short, yeast containing TAP tags on TFIIH subunits Tfb4 and Ssl2 was grown in 100 liters of YPAD (yeast extract, peptone, adenine, glucose) medium to an optical density (OD) of 10.0. Whole cell lysate was prepared by bead beating in buffer A [50 mM Hepes (pH 7.6), 1 mM EDTA, 5% glycerol, 400 mM potassium acetate, 2-mercaptoethanol, and protease inhibitors]. Following the addition of 100 mM ammonium sulfate and 0.1% polyethyleneimine (PEI), lysed cells were stirred for 1 hour and centrifuged, and then the cleared lysate was loaded onto an immunoglobulin G (IgG) column. The column was washed with 5 to 10 column volumes of buffer 300 [50 mM Hepes (pH 7.6), 1 mM EDTA, 5% glycerol, 300 mM potassium acetate, 2 mM dithiothreitol (DTT), and protease inhibitors] and then resuspended in buffer 300 and allowed to settle. IgG beads were washed by batch with another 10 column volumes of buffer 300. TFIIH was treated with tobacco etch virus in buffer 300, eluted from the IgG column, and loaded onto a UnoQ column (Bio-Rad). TFIIH was eluted by salt gradient of concentration from 300 mM to 1.2 M potassium acetate. Fractions containing different TFIIH subunits were separated and concentrated separately.