Jun 04, 2025
Cycloheximide assay (Thermo Fisher Scientific, Catalog #AC357420010)
- 1NIMH
Protocol Citation: Jailyn Izu 2025. Cycloheximide assay (Thermo Fisher Scientific, Catalog #AC357420010). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92mb4l3e/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: April 17, 2024
Last Modified: March 26, 2026
Protocol Integer ID: 98346
Keywords: life of tbk1 n455, tbk1 n455, type tbk1, thermo fisher scientific, assay
Funders Acknowledgements:
National Institute of Mental Health
Grant ID: ZIA MH002982
National Institute of Mental Health
Grant ID: R01MH122471
National Institute of Neurological Disorders and Stroke
Grant ID: U01NS120836
Foundation for the National Institutes of Health
Grant ID: Deeda Blair Research Initiative for Disorders of the Brain
Abstract
Assay used to evaluate the half-life of TBK1 N455S as compared to wild-type TBK1.
Materials
- COS-1 cells
- EGFP plasmid (OmicsLink‱ Expression Clone EX-EGFP-M11; GeneCopoeia, Inc., Rockville, MD)
- Flag-tagged TBK1 expression plasmid (OmicsLink‱ Expression Clone EX-U0617-M11; GeneCopoeia, Inc., Rockville, MD)
- HA-tagged TBK1 expression plasmid (OmicsLink‱ Expression Clone EX-U0617-M09; GeneCopoeia, Inc., Rockville, MD)
- Cycloheximide (Thermo Fisher Scientific, Catalog #AC357420010)
- Dimethyl Sulfoxide (DMSO) ()
- M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Catalog #78505)
- Halt Protease Inhibitor Cocktail, EDTA-free (100X) (Thermo Fisher Scientific, Catalog #78425)
- 6-well plate (Corning, #3506)
- FuGene 4K Transfection Reagent (Promega, #E5911)
- Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific, #31985062)
- Plasmid constructs
- Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMax (Thermo Fisher Scientific, #10569010)
- FBS (Gibco, #26140-079)
- Penicillin-streptomycin (Thermo Fisher Scientific, #15140122)
- PBS, pH 7.4 (Thermo Fisher Scientific, #10010023)
- M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, #78501)
- Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, #78429)
Seed cells
Seed 100,000 COS-1 cells per well (2 mL total volume per well) in a 6-well plate. Do not add CHX yet. Incubate the plated cells in 37 °C with 5% CO2 for 48 hours.
Transfection
Follow Steps 2-6 in the general protocol for transfection:
Protocol
CREATED BY
Jailyn Izu
Protocol-specific parameters:
- For Step 2, use a 1:3 DNA-to-FuGENE (μg/μL) ratio, with 2 μg of total DNA for each sample.
- For Step 3, use the Flag-TBK1 plasmid constructs from "Flag-TBK1 N455S and Flag-TBK1 M559R cloning" protocol and co-transfect with EGFP (OmicsLink‱ Expression Clone EX-EGFP-M11; GeneCopoeia, Inc., Rockville, MD)
- For Step 6, incubate transfected cells for 24 hours 24:00:00 .
1d
Treat with cycloheximide
8h
Dilute cycloheximide (CHX) in DMSO to a final concentration of 10 mg/mL .
Add 20 µL of 10 mg/mL CHX (for a final concentration in the wells of 100 ug/ml CHX ) or 20 µL DMSO (for controls) to the 24-hour time point wells only.
Note the time added.
After 08:00:00 add 20 µL of 10 mg/mL CHX (for a final concentration of 100 ug/ml ) or 20 µL of DMSO to the 16 hour time point wells only.
Note the time added.
8h
8 hours later, end the time course by follow Steps 7-12 in the general protocol for transfection:
Protocol
CREATED BY
Jailyn Izu
Protocol-specific parameters:
- For Step 9, add 300 µL / well M-PER reagent + 1X protease and phosphatase inhibitor to each well