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Cell culture, transfection and labeling of HaloTag-Ras
Last updated date: May 2, 2025 Views: 19 Forks: 0
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On the day of imaging, coverslips were washed with phosphate buffer saline and cells were labeled with 25pM fluorescent (JF646 or JF549) HaloTag ligand (30 minutes), which covalently binds to the HaloTag-RAS molecules. After incubation with the ligand, cells are washed three times with PBS before replenishing cells with complete medium. It is a good idea to use complete medium without phenol red, as this can also contribute to background.
Copyright: © 2025 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Turbyville, T(2025). Cell culture, transfection and labeling of HaloTag-Ras. Bio-protocol Preprint. bio-protocol.org/prep2811.
- Goswami, D., Chen, D., Yang, Y., Gudla, P. R., Columbus, J., Worthy, K., Rigby, M., Wheeler, M., Mukhopadhyay, S., Powell, K., Burgan, W., Wall, V., Esposito, D., Simanshu, D. K., Lightstone, F. C., Nissley, D. V., McCormick, F. and Turbyville, T.(2020). Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior. eLife. DOI: 10.7554/eLife.47654
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