Purification of human brain metallothionein by organic and reversed-phase high-performance liquid chromatography under acidic conditions.
A simplified high-performance liquid chromatographic method for the detection of metallothioneins, notably metallothionein-III, has been developed. In order to purify metallothionein, differential acetone precipitation at 50% (v/v) and at 80% (v/v) was employed on a 20% normal human brain homogenate. The reconstituted pellet was injected into a C18 microbore reversed-phase HPLC column, equilibrated with 0.1% trifluoroacetic acid, and developed at a flow-rate of 800 microliter/min with a linear gradient from 0% to 60% acetonitrile in 0.094% trifluoroacetic acid for 60 min. Western blots indicated that metallothioneins-I and II eluted at 16% acetonitrile and metallothionein-III eluted at 37% acetonitrile.
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Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
D000595:Amino Acid Sequence; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008668:Metallothionein / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000302:isolation & purification; D016622:Silver Staining
D009419:Nerve Tissue Proteins; D008668:Metallothionein