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: Biochem J

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

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-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 ( Pt 2)
Publication statusPublished - 1992

Keywords

  • Affinity Labels Amino Acid Sequence Animals Autoradiography *Bacterial Proteins *Biotin Detergents Electrophoresis, Polyacrylamide Gel Magnetics Melanoma Mice Microspheres Molecular Sequence Data Octoxynol Photochemistry Polyethylene Glycols Receptors, Pituitary Hormone/*isolation & purification/metabolism Streptavidin Tumor Cells, Cultured alpha-MSH/*metabolism

Cite this

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title = "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: Biochem J",
abstract = "The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-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.",
keywords = "Affinity Labels Amino Acid Sequence Animals Autoradiography *Bacterial Proteins *Biotin Detergents Electrophoresis, Polyacrylamide Gel Magnetics Melanoma Mice Microspheres Molecular Sequence Data Octoxynol Photochemistry Polyethylene Glycols Receptors, Pituitary Hormone/*isolation & purification/metabolism Streptavidin Tumor Cells, Cultured alpha-MSH/*metabolism",
author = "Ahmed, {A. R.} and Olivier, {G. W.} and G. Adams and Erskine, {M. E.} and Kinsman, {R. G.} and Branch, {S. K.} and Moss, {S. H.} and Notarianni, {L. J.} and Pouton, {C. W.}",
note = "Ahmed, A R Olivier, G W Adams, G Erskine, M E Kinsman, R G Branch, S K Moss, S H Notarianni, L J Pouton, C W eng Research Support, Non-U.S. Gov't ENGLAND 1992/09/01 Biochem J. 1992 Sep 1;286 ( Pt 2):377-82.",
year = "1992",
language = "English",
volume = "286 ( Pt 2)",
pages = "377--382",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press",

}

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

In: Biochemical Journal, Vol. 286 ( Pt 2), 1992, p. 377-382.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - 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

T2 - Biochem J

AU - Ahmed, A. R.

AU - Olivier, G. W.

AU - Adams, G.

AU - Erskine, M. E.

AU - Kinsman, R. G.

AU - Branch, S. K.

AU - Moss, S. H.

AU - Notarianni, L. J.

AU - Pouton, C. W.

N1 - Ahmed, A R Olivier, G W Adams, G Erskine, M E Kinsman, R G Branch, S K Moss, S H Notarianni, L J Pouton, C W eng Research Support, Non-U.S. Gov't ENGLAND 1992/09/01 Biochem J. 1992 Sep 1;286 ( Pt 2):377-82.

PY - 1992

Y1 - 1992

N2 - The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-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.

AB - The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-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.

KW - Affinity Labels Amino Acid Sequence Animals Autoradiography Bacterial Proteins Biotin Detergents Electrophoresis, Polyacrylamide Gel Magnetics Melanoma Mice Microspheres Molecular Sequence Data Octoxynol Photochemistry Polyethylene Glycols Receptors, Pitu

M3 - Article

VL - 286 ( Pt 2)

SP - 377

EP - 382

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -