期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 128, 期 -, 页码 101-108出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2016.08.011
关键词
CRISPR-mediated purification; Integrator complex; Mass spectrometry
类别
资金
- Cancer Prevention and Research Institute of Texas [RP140800]
- Welch Foundation [H1889]
- Department of Biochemistry and Molecular Biology at UTMB
The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT. (C) 2016 Elsevier Inc. All rights reserved.
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