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

Maria L. Sorkin, Dmitri A. Nusinow

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Scopus citations

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages189-203
Number of pages15
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2398
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Affinity purification
  • Circadian clock
  • Mass spectrometry
  • Protein-protein interactions

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