Immunoprecipitation of plasma membrane receptor-like kinases for identification of phosphorylation sites and associated proteins Yasuhiro Kadota1, Alberto P. Macho2 and Cyril Zipfel The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK 1Present address: RIKEN Center for Sustainable Resource Science, Plant Immunity Research Group, Suehiro-cho 1-7-22 Tsurumi-ku, Yokohama 230-0045, Japan. 2Present address: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai 201602, China. Summary Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilisation, purification, identification of associated proteins and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins and to identify modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. Keywords: immunoprecipitation, protein phosphorylation, receptor-like kinase Introduction The plasma membrane separates the cytoplasm of plant cells from the outside environment. Therefore, there are numerous receptors recognizing a variety of environmental signals at the plasma membrane. Receptor-like kinases (RLKs) are a
Immunoprecipitation of plasma membrane receptor-like kinases for identification of phosphorylation sites and associated proteins Yasuhiro Kadota1, Alberto P. Macho2 and Cyril Zipfel The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK 1Present address: RIKEN Center for Sustainable Resource Science, Plant Immunity Research Group, Suehiro-cho 1-7-22 Tsurumi-ku, Yokohama 230-0045, Japan. 2Present address: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai 201602, China. Summary Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilisation, purification, identification of associated proteins and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins and to identify modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. Keywords: immunoprecipitation, protein phosphorylation, receptor-like kinase Introduction The plasma membrane separates the cytoplasm of plant cells from the outside environment. Therefore, there are numerous receptors recognizing a variety of environmental signals at the plasma membrane. Receptor-like kinases (RLKs) are a