- Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis.
Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis.
Methods in molecular biology (Clifton, N.J.) (2021-10-22)
Maria L Sorkin, Dmitri A Nusinow
PMID34674177
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.
MATERIALS
Product Number
Brand
Product Description
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)