Biochemical purification of native immune protein complexes

Methods Mol Biol. 2011:712:31-44. doi: 10.1007/978-1-61737-998-7_4.

Abstract

Protein complex purification represents a powerful approach to identify novel players in plant innate immunity. However, the identification of interacting protein partners within a natural context has been a challenge for researchers. In this chapter, we describe a method of immunoaffinity chromatography using purified, antibodies to isolate native protein complexes from wild-type tissue. We detail the antibody purification and immobilization steps in addition to the co-immunoprecipitation protocol. In addition, a method to prepare protein samples for mass spectroscopy analysis is described. This straightforward protocol has been used to isolate and identify novel components of Arabidopsis immunity-associated protein complexes.

MeSH terms

  • Antigen-Antibody Complex / isolation & purification*
  • Arabidopsis / chemistry
  • Arabidopsis / immunology
  • Arabidopsis Proteins / isolation & purification*
  • Chromatography, Affinity / instrumentation
  • Chromatography, Affinity / methods*
  • Immunoprecipitation / instrumentation
  • Immunoprecipitation / methods*
  • Plant Immunity / immunology

Substances

  • Antigen-Antibody Complex
  • Arabidopsis Proteins