Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads

A. R.H. Ahmed, G. W.J. Olivier, G. Adams, M. E. Erskine, R. G. Kinsman, S. K. Branch, S. H. Moss, L. J. Notarianni, C. W. Pouton

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29 Citations (Scopus)

Abstract

The α-melanocyte-stimulating hormone (α-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of α-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptavidin/biotin-based affinity system was used to isolate the α-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled α-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the α-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.

Original languageEnglish
Pages (from-to)377-382
Number of pages6
JournalBiochemical Journal
Volume286
Issue number2
DOIs
Publication statusPublished - 1 Jan 1992

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