Jun 04, 2025
MG-132 (Invitrogen, #tlrl-mg132-2) and hydroxychloroquine (Sigma, #H0915-5MG) assay
- 1NIMH
Protocol Citation: Jailyn Izu 2025. MG-132 (Invitrogen, #tlrl-mg132-2) and hydroxychloroquine (Sigma, #H0915-5MG) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokrpxl4o/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: May 15, 2024
Last Modified: March 26, 2026
Protocol Integer ID: 99888
Keywords: role for various protein degradation pathway, various protein degradation pathway, tbk1 n455, hydroxychloroquine, tbk1, mg132, 5mg
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
Abstract
Protocol for evaluating the role for various protein degradation pathways on the half-life of TBK1 N455S
Materials
- COS-1 cells
- Cycloheximide (Thermo Fisher Scientific, Catalog #AC357420010)
- Dimethyl Sulfoxide (DMSO), filtered ()
- RNAse free H2O, filter (Quality biological, #351-029-721)
- M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Catalog #78505)
- Halt Protease Inhibitor Cocktail, EDTA-free (100X) (Thermo Fisher Scientific, Catalog #78425)
- MG-132 (Invitrogen, #tlrl-mg132-2)
- Hydroxychloroquine (Sigma, #H0915-5MG)
Create stock solutions of MG-132 and HCQ
Obtain 5 mg MG-132 from Invitrogen (#tlrl-mg132-2)
Reconstitute MG-132 with 250 µL DMSO (filtered) to a final concentration of 42 millimolar (mM) .
Create 25 10 µL aliquots and store in -20 °C . These are your stock solutions which are stable for up to 3 months. You will dilute these aliquots for the experiment.
Obtain 5 mg hydroxychloroquine sulfate (HCQ) from Sigma (#H0915-5MG)
Reconstitute HCQ with 250 µL RNAse free H2O (filtered) to a final concentration of 46 millimolar (mM) .
Create 25 10 µL aliquots and store in -20 °C . These are your stock solutions. You will dilute these aliquots for the experiment.
Prepare MG-132 and HCQ dilutions for the experiment
For MG-132, create a 1:21 dilution of 42 mM stock solution in media to obtain a 2 millimolar (mM) dilution. You will add a volume of this to the cells at each time point.
Perform dilutions needed for the experiment the day of, as dilutions of the stock solution should not be stored for more than 24 hours.
For HCQ, create a 1:21 dilution of 46 mM stock solution in H2O to obtain a 5 millimolar (mM) dilution. You will add a volume of this to the cells at each time point.
Perform dilutions needed for the experiment the day of, as dilutions of the stock solution should not be stored for more than 24 hours.
Perform cycloheximide assay
Follow the protocol for the cycloheximide assay, treating with CHX + MG-132 OR HCQ at time points (READ PROTOCOL-SPECIFIC PARAMETERS BELOW BEFORE BEGINNING).
Protocol
CREATED BY
Christopher Bartley
Protocol-specific parameters:
There will be 3 conditions: (1) CHX alone, (2) CHX + MG132, and (3) CHX + HCQ). Therefore,
- For Step 4 you will,
(1) Add 20 µL of 10 mg/mL CHX (for a final concentration in the wells of 100 ug/ml CHX) + 20 µL H2O (filtered) to the 24-hour time point, CHX alone wells only.
(2) add 20 µL of 10 mg/mL CHX + 20 µL of 2 millimolar (mM) MG-132 to the 24-hour time point, CHX + MG-132 wells only.
(3) add 20 µL of 10 mg/mL CHX + 20 µL of 5 millimolar (mM) HCQ to the 24-hour time point, CHX + HCQ wells only.
- For Step 5 you will,
(1) Add 20 µL of 10 mg/mL CHX (for a final concentration of 10 ug/ml CHX) + 20 µL H2O to the 16-hour time point, CHX alone wells only.
(2) add 20 µL of 10 mg/mL CHX + 20 µL of 2 millimolar (mM) MG-132 to the 16-hour time point, CHX + MG-132 wells only.
(3) add 20 µL of 10 mg/mL CHX + 20 µL of 5 millimolar (mM) HCQ to the 16-hour time point, CHX + HCQ wells only.