Detection of Metallothionein (MT) Proteins with Radiolabeled [14C]Iodoacetamide

D. R. Conklin, K. S. Cowan, M. Aschner

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Carboxymethylation of protein samples prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is known to improve the transfer and detection of metallothioneins (MTs) on nitrocellulose membranes by Western blotting. Dissociation of metals from MTs by denaturing the samples with SDS, reduction of sulfhydryl (-SH) residues on MT-associated cysteines with dithiothreitol (DTT), and subsequent Carboxymethylation of the cysteine residues with iodoacetamide overcome disulfide bond formation between MT molecules, preventing their aggregation, and allow for the electrophoretic transfer and detection of MTs by Western blotting. In the present study, advantage is taken of cysteine residue bond formation by the addition of 14C-radiolabeled iodoacetamide to the Carboxymethylation process to validate β-emission autoradiography as an alternative method to Western blotting. The analysis of MTs by β-emission autoradiography suggests that treatment of neonatal rat primary astrocyte cultures with cadmium chloride (CdCl2), a potent MT inducer, leads to an increase in protein levels that is restricted and identical to the migratory pattern of MTs on Western blots. β-Emission autoradiography affords a new, reproducible, and inexpensive alternative method for the detection of MTs.

Original languageEnglish (US)
Pages (from-to)149-155
Number of pages7
JournalToxicology Methods
Volume6
Issue number3
DOIs
StatePublished - Jan 1 1996
Externally publishedYes

Keywords

  • Astrocyte
  • Cadmium
  • Carboxymethylation
  • Metallothionein
  • Method
  • β-autoradiography

ASJC Scopus subject areas

  • Toxicology

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