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. Author manuscript; available in PMC: 2010 Apr 28.
Published in final edited form as: FASEB J. 2007 Oct 30;22(3):721–732. doi: 10.1096/fj.07-9199com

Figure 5.

Figure 5

Expression, purification, and labeling of recombinant hexahistidine-tagged HMGCS. A) Protein (10 μg) from each purification step was separated on a 10% SDS-PAGE as described in Materials and Methods. Lane 1: total bacterial lysate. Lane 2: S10 supernatant of a 10,000 g centrifugation. Lane 3: elution from the Ni-chelating column. Lane 4: elution from anion exchange column. Lane 5: concentrated protein after ultrafiltration with Centricon Plus-20. B) Recombinant HMGCS-His6 (1 μg) labeled with 1 μCi of [125I]iodopalmitoyl-CoA ([125I]IC16-CoA). Acylation was detected by autoradiography (left panel), and proteins by staining with Coomassie blue (right panel). C) HMGCS-His6 samples (1 μg) were labeled with azido-palmitoyl-CoA (N3C14-CoA) with or without reaction with phosphine-Myc. Acylated protein was detected by Western blot with anti-Myc antibody (left panel), and corresponding proteins were stained with Coomassie blue (right panel). D) HMGCS-His6 samples (1 μg) were labeled with azido-palmitoyl-CoA (N3C14-CoA) and reacted with or without phosphine-biotin. Acylated protein was detected by Western blotting with Neutravidin-HRP/ECL (left panel) and corresponding proteins were stained with Coomassie blue (right panel).