sentence: It was purified as TM68 except that after cell lysis and ultracentrifugation step to isolate a crude membrane fraction , soluble supernatant fraction was used to purify Ubc6TM with the same buffers without detergent.Uba1 , Ubc7 , Cue1 , Cdc48 , Npl4-Ufd1 were purified as described ( Stein et al ., 2014 ) .