TurboID Labeling and Analysis of Proteins in the Primary Cilium
Known as the cell’s antenna and signaling hub, the primary cilium is a hair-like organelle with a few micrometers in length and 200–300 nm in diameter. Due to the small size of the primary cilium, it is technically challenging to profile ciliary proteins from mammalian cells. Traditional methods, su...
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Bio-protocol LLC
2025-05-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5303&type=0 |
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| author | Xiaoliang Liu Xuecai Ge |
| author_facet | Xiaoliang Liu Xuecai Ge |
| author_sort | Xiaoliang Liu |
| collection | DOAJ |
| description | Known as the cell’s antenna and signaling hub, the primary cilium is a hair-like organelle with a few micrometers in length and 200–300 nm in diameter. Due to the small size of the primary cilium, it is technically challenging to profile ciliary proteins from mammalian cells. Traditional methods, such as physical isolation of cilia, are susceptible to contamination from other cellular components. Other proximity-based labeling methods via APEX or BioID have been used to map ciliary proteins. However, these approaches have their inherent limitations, including the use of toxic reagents like H2O2 and prolonged labeling kinetics. Here, we show a new proximity-based labeling technique for primary cilia with TurboID. TurboID presents a distinct advantage over BioID and APEX2 due to its expedited labeling kinetics, taking minutes instead of hours, and its use of a non-toxic biotin substrate, which eliminates the need for H2O2. When targeted to the cilium, TurboID selectively labels ciliary proteins with biotin. The biotinylated proteins are then enriched with streptavidin beads and labeled with tandem mass tags (TMT), followed by mass spectrometry (MS) detection. This protocol eliminates the requirement of toxic labeling reagents and significantly reduces the labeling time, thus providing advantages in mapping signaling proteins with high temporal resolution in live cells. |
| format | Article |
| id | doaj-art-60ef315caec042239aba3f3219638b5e |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-60ef315caec042239aba3f3219638b5e2025-08-20T03:48:46ZengBio-protocol LLCBio-Protocol2331-83252025-05-0115910.21769/BioProtoc.5303TurboID Labeling and Analysis of Proteins in the Primary CiliumXiaoliang Liu0Xuecai Ge1Department of Molecular and Cell Biology, University of California, Merced, Merced California, CA, USADepartment of Molecular and Cell Biology, University of California, Merced, Merced California, CA, USAKnown as the cell’s antenna and signaling hub, the primary cilium is a hair-like organelle with a few micrometers in length and 200–300 nm in diameter. Due to the small size of the primary cilium, it is technically challenging to profile ciliary proteins from mammalian cells. Traditional methods, such as physical isolation of cilia, are susceptible to contamination from other cellular components. Other proximity-based labeling methods via APEX or BioID have been used to map ciliary proteins. However, these approaches have their inherent limitations, including the use of toxic reagents like H2O2 and prolonged labeling kinetics. Here, we show a new proximity-based labeling technique for primary cilia with TurboID. TurboID presents a distinct advantage over BioID and APEX2 due to its expedited labeling kinetics, taking minutes instead of hours, and its use of a non-toxic biotin substrate, which eliminates the need for H2O2. When targeted to the cilium, TurboID selectively labels ciliary proteins with biotin. The biotinylated proteins are then enriched with streptavidin beads and labeled with tandem mass tags (TMT), followed by mass spectrometry (MS) detection. This protocol eliminates the requirement of toxic labeling reagents and significantly reduces the labeling time, thus providing advantages in mapping signaling proteins with high temporal resolution in live cells.https://bio-protocol.org/en/bpdetail?id=5303&type=0 |
| spellingShingle | Xiaoliang Liu Xuecai Ge TurboID Labeling and Analysis of Proteins in the Primary Cilium Bio-Protocol |
| title | TurboID Labeling and Analysis of Proteins in the Primary Cilium |
| title_full | TurboID Labeling and Analysis of Proteins in the Primary Cilium |
| title_fullStr | TurboID Labeling and Analysis of Proteins in the Primary Cilium |
| title_full_unstemmed | TurboID Labeling and Analysis of Proteins in the Primary Cilium |
| title_short | TurboID Labeling and Analysis of Proteins in the Primary Cilium |
| title_sort | turboid labeling and analysis of proteins in the primary cilium |
| url | https://bio-protocol.org/en/bpdetail?id=5303&type=0 |
| work_keys_str_mv | AT xiaoliangliu turboidlabelingandanalysisofproteinsintheprimarycilium AT xuecaige turboidlabelingandanalysisofproteinsintheprimarycilium |