A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells
The Mediator, a multi-subunit protein complex in all eukaryotes, comprises the core mediator (cMED) and the CDK8 kinase module (CKM). As a molecular bridge between transcription factors (TFs) and RNA polymerase II (Pol II), the Mediator plays a critical role in regulating Pol II–dependent transcript...
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Bio-protocol LLC
2025-02-01
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| author | Hui- Tang Kuang- Tsai Ti- Chao |
| author_facet | Hui- Tang Kuang- Tsai Ti- Chao |
| author_sort | Hui- Tang |
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| description | The Mediator, a multi-subunit protein complex in all eukaryotes, comprises the core mediator (cMED) and the CDK8 kinase module (CKM). As a molecular bridge between transcription factors (TFs) and RNA polymerase II (Pol II), the Mediator plays a critical role in regulating Pol II–dependent transcription. Considering its large size and complex composition, conducting in vitro studies on the Mediator complex is challenging, especially when isolating the intact and homogeneous complex from human cells. Here, we present a method to purify the intact CKM-cMED complex from FreeStyle 293-F cells (293-F cells), which offers advantages for performing large-scale protein purification. To isolate the CKM-bound cMED without the presence of Pol II, FLAG-tagged CDK8, a subunit of the CKM complex, was expressed in 293-F cells for purification, as CKM and Pol II are mutually exclusive in their interaction with cMED. The complex is isolated from nuclear extracts through immunoaffinity purification and further purified by glycerol gradient to enhance its homogeneity. This protocol provides a time- and cost-efficient way to purify the endogenous Mediator complex for structural- and functional-based studies. |
| format | Article |
| id | doaj-art-fd59ba6b35f1459fb3245c9bef07949d |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-fd59ba6b35f1459fb3245c9bef07949d2025-08-20T02:02:05ZengBio-protocol LLCBio-Protocol2331-83252025-02-0115410.21769/BioProtoc.5185A Protocol to Purify Human Mediator Complex From Freestyle 293-F CellsHui- Tang0Kuang- Tsai1Ti- Chao2Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USADepartment of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USADepartment of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USAThe Mediator, a multi-subunit protein complex in all eukaryotes, comprises the core mediator (cMED) and the CDK8 kinase module (CKM). As a molecular bridge between transcription factors (TFs) and RNA polymerase II (Pol II), the Mediator plays a critical role in regulating Pol II–dependent transcription. Considering its large size and complex composition, conducting in vitro studies on the Mediator complex is challenging, especially when isolating the intact and homogeneous complex from human cells. Here, we present a method to purify the intact CKM-cMED complex from FreeStyle 293-F cells (293-F cells), which offers advantages for performing large-scale protein purification. To isolate the CKM-bound cMED without the presence of Pol II, FLAG-tagged CDK8, a subunit of the CKM complex, was expressed in 293-F cells for purification, as CKM and Pol II are mutually exclusive in their interaction with cMED. The complex is isolated from nuclear extracts through immunoaffinity purification and further purified by glycerol gradient to enhance its homogeneity. This protocol provides a time- and cost-efficient way to purify the endogenous Mediator complex for structural- and functional-based studies.https://bio-protocol.org/en/bpdetail?id=5185&type=0 |
| spellingShingle | Hui- Tang Kuang- Tsai Ti- Chao A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells Bio-Protocol |
| title | A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells |
| title_full | A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells |
| title_fullStr | A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells |
| title_full_unstemmed | A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells |
| title_short | A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells |
| title_sort | protocol to purify human mediator complex from freestyle 293 f cells |
| url | https://bio-protocol.org/en/bpdetail?id=5185&type=0 |
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