Cdc48A pulldown from an amino lonk column baited with SAMP1

H. volcanii Cdc48a, RecJ3/4 and RNase J associate with Ubl-coated beads. A) Strategy to isolate H. volcanii proteins that bind agarose beads charged with monomeric Ubl (vs. BSA) as bait. Cell lysate was from triplicate cultures of H. volcanii NH02-pJAM957. B) Left, Non-reducing SDS-PAGE of H. volcanii proteins that bound BSA- (control, lane 1) vs. Ubl- (lane 2) decorated beads in the presence of ATP. Gels were stained with SYPRO Ruby. Red bar, HMW(250kDa) and LMW(50kDa) (high and low molecular weight) regions of gel excised from lanes 1 and 2 for LC- MS/MS analysis. Right, Proteins identified at > 99.9% probability and < 0.1 % false discovery rate (FDR) in the Ubl (not BSA) samples. Similar trend was observed in independent experiment. ATP was required for this association. Theor. Mr, theoretical molecular mass based on genome sequence. Normalized total spectra of sample minus control is indicated.