Protocol

Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis

Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark

¹ Present address: Carlsberg Laboratory, 2500 Valby, Denmark

² Corresponding author (mundy{at}bio.ku.dk)

INTRODUCTION

Transcriptional reprogramming occurs during development and in response to diverse stimuli and stresses. The isolation and characterization of nuclear proteins, particularly those binding to DNA and chromatin, are therefore important to understanding these processes. Two specific approaches to understanding the function of nuclear proteins involve the characterization of their protein-protein interactions, and of the transcriptional targets of specific transcription factors. Coimmunoprecipitation (co-IP) is a straightforward technique to study in vivo protein-protein interactions, and can identify interacting proteins or protein complexes present in cell extracts. Chromatin immunoprecipitation (ChIP) permits the identification of protein-DNA interactions in pull-down assays using specific antibodies against DNA-binding proteins, such as transcription factors or histone/chromatin-binding proteins. Here, we present detailed protocols for extraction of Arabidopsis seedlings, co-IP of nuclear proteins, and ChIP.