Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis.
| Autor: | Sorkin ML; Donald Danforth Plant Science Center, St. Louis, MO, USA.; Division of Biology and Biomedical Sciences, Washington University in St. Louis, St. Louis, MO, USA., Nusinow DA; Donald Danforth Plant Science Center, St. Louis, MO, USA. meter@danforthcenter.org. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2398, pp. 189-203. |
| DOI: | 10.1007/978-1-0716-1912-4_15 |
| Abstrakt: | 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. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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