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Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein

Planta volume 176, pages 519–526 (1988)Cite this article

Abstract

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.

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Abbreviations

ABP:

auxin-binding protein

DEAE:

diethylaminoethyl

Ig:

immunoglobulin

kDa:

kilodalton

NAA:

naphthalene-1-acetic acid

Mr :

relative molecular mass

PAGE:

polyacrylamide gel electrophoresis

SDS:

sodium dodecyl sulfate

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Affiliations

  1. AFRC Institute of Horticultural Research, East Malling, ME19 6BJ, Maidstone, Kent

    Richard M. Napier, Michael A. Venis & Michael A. Bolton

  2. AFRC Institute of Animal Physiology and Genetics Research, Cambridge Research Station, Monoelonal Antibody Centre, Babraham, CB2 4AT, Cambridge, UK

    Louise I. Richardson & Goeffrey W. Butcher

Authors
  1. Richard M. Napier
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  2. Michael A. Venis
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  3. Michael A. Bolton
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  4. Louise I. Richardson
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  5. Goeffrey W. Butcher
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Napier, R.M., Venis, M.A., Bolton, M.A. et al. Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein. Planta 176, 519–526 (1988). https://doi.org/10.1007/BF00397659

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  • DOI: https://doi.org/10.1007/BF00397659

Key words

  • Antibody (auxin)
  • Auxin (receptor, antibody)
  • Zea (auxin-binding protein)