Sep 23, 2023
Rapalog-induced chemical dimerization experiments
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2023. Rapalog-induced chemical dimerization experiments. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmyynl5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84159
Keywords: Rapalog-induced chemical dimerization experiments, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes Rapalog-induced chemical dimerization experiments.
Attachments
760-1928.pdf
35KB
Materials
Reagents
iDimerize™ Inducible Heterodimer SystemTakara Bio Inc.Catalog #635067
LSR Fortessa Cell Analyzer (BD Biosciences)
Rapalog-induced chemical dimerization experiments
Rapalog-induced chemical dimerization experiments
1d
1d
To perform chemical-induced dimerization (CID) experiments we will first establish cell lines expressing the FRB-Fis1 and FKBP-GFP-GOI (FKBP-GFP fused to our gene of interest).
HeLa cells are consecutively transduced with lentivirus for FRB-Fis1, which also expresses mitochondrially targeted monoKeima (mt-mKeima), and then with FKBP-GFP-GOI. See the lentivirus transduction protocol for further details.
Once cell lines are established, cells are seeded in 6-well plates the day before the experiment (density: 800k per well).
Induce FKBP-FRB dimerisation by treating the cells with 50 nanomolar (nM) Rapalog A/C hetero-dimerizer rapalog (635057, Takara) for 24:00:00 .
1d
After 24h, collect cells by trypsinization and take the cells to the FACS facility for mt-mKeima analysis.
Analyze the cells by flow cytometry, using the mt-mKeima ratio (561/405) as a readout with an LSR Fortessa Cell Analyzer (BD Biosciences).
Gate the cells for GFP/mt-mKeima double-positive cells, and collect 10,000 events for this population.
Analyze data using FlowJo.