Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis

Identification of protein-protein interactions is an effective method of elucidating new roles for circadian clock-associated proteins that can expand beyond the information collected from transcriptional studies and genetic screens. Tandem affinity purification coupled with liquid chromatography mass spectrometry (APMS) utilizes epitope-tagged versions of your protein of interest to co-precipitate direct and indirect protein partners. Here, we provide a protocol and suggestions for proper design of 6x-His-3x-FLAG-tagged clock proteins and isolation of protein-protein interactions using two immunoprecipitation steps for increased specificity.