Figure 3.

SDS-PAGE of human HMGCL under reducing or nonreducing conditions. Purified HMGCL was subjected to dialysis (>16 hr) in air equilibrated buffer. An aliquot was incubated with the sulfhydryl-specific alkylating reagent NEM (lanes 2, 5) while another aliquot was mock treated (lanes 1, 4). Reactions were split in half and denatured in an equal volume of SDS loading buffer and treated with (lanes 1-3) or without (lanes 4-5) mercaptoethanol (2%) prior to heating (95 °C) for 5 min. Five ug of protein was loaded in each lane of a 12% SDS-PAGE gel. Molecular weight markers (lane 3) include: phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21,5 kDa; lysozyme, 14.4 kDa.