Nov 20, 2024
L-HPG Metabolic labeling of mitochondrial translation in cultured cell lines
- 1UC Berkeley
Protocol Citation: Adam Begeman, Samantha C Lewis 2024. L-HPG Metabolic labeling of mitochondrial translation in cultured cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wedrvo5/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
This is our working protocol as of 11/18/2024.
Created: October 23, 2024
Last Modified: November 20, 2024
Protocol Integer ID: 110941
Keywords: L-HPG, Immunofluorescence, translation, mitochondria, mitoribosome, click chemistry
Funders Acknowledgement:
NIH
Grant ID: R35GM147218
Abstract
This protocol describes the metabolic labeling of mitochondrial translation in cultured cells (such as IMR90) using an amino acid L-Homopropargylglycine (L-HPG).
Guidelines
Protocol Notes:
- 15 min, 30 min, and 1 hr incubation times have been used
5 minutes has been published
- Can click with either 647 or 488 azide
- This protocol is NOT compatible with glutaraldehyde fixation due to cytoplasmic signal drowning out mitochondrial
Materials
Buffers and Reagents:
- Fixative: 4% PFA in DPBS (lacking Calcium and Magnesium), pH 7.4
- Quenching Solution: 1M Glycine in DPBS
- Permeabilization Solution: DPBS + 0.1% Triton-X 100
- Blocking/ Staining Solution: TBST + 1% BSA
- DPBS
- Methionine Free DMEM (https://www.thermofisher.com/order/catalog/product/21013024)
- 50 mg/mL Cycloheximide (1000x)
- 50 mg/mL Chloramphenicol (1000x)
- L-HPG (50 mM) (1000x) (https://www.thermofisher.com/order/catalog/product/C10186?SID=srch-srp-C10186)
- Click Reaction Cocktail (Made Fresh Right Before Use)
Cultured cell line
Note
Following volumes are for a single 35 mm imaging dish
| A | B | |
| Reaction buffer | 440 uL | |
| Copper protectant solution | 10 uL | |
| Fluorescent azide | 1.2 uL | |
| Click reaction additive solution (1:10 solution in water of stock additive) | 50 uL |
Procedure
Procedure
1h 57m
1h 57m
Remove media from adherent IMR90 cells cultured in 35 mm cell culture dish and wash once with DPBS pre-warmed to 37 °C .
Replace DPBS with 1 mL methionine free DMEM pre warmed to 37 °C .
Incubate at 37 °C for 00:10:00 .
10m
Add to cell culture dish Cycloheximide to a final concentration of 50 μg/ml (1:1000) e.g. 1 microliter into 1 mL of Methionine-free medium.
Note
Add Chloramphenicol to a final concentration of 50 µg per ml (1:1000) for mitochondrial translation inhibition control.
Incubate at 37 °C for 00:20:00 .
20m
Add L-HPG to dish to a final concentration of 50 micromolar (µM) (1:1000 dilution into 1 mL ) and incubate at 37 °C for desired amount of time.
Remove media and replace with methionine free DMEM pre warmed to 37 °C .
Incubate at 37 °C for 00:05:00 .
5m
Remove media from cell culture dish and immediately cover the dish with 2 mL fixative solution pre warmed to 37 °C .
Incubate at Room temperature for 00:30:00 .
30m
Quench fixation with 200 µL of quenching solution (00:02:00 ).
2m
Wash 1x with DPBS.
Remove DPBS and cover the dish with 2 mL of permeabilization solution and incubate at Room temperature for 00:20:00 .
20m
Wash 1X with DPBS.
Remove DPBS and add 500 µL click reaction cocktail and incubate at Room temperature in the dark for 00:30:00 .
Note
Click reaction cocktail should be made fresh in the order above directly before use.
30m
Add 1.5 mL blocking solution to dish and then aspirate off blocking solution/ click cocktail mixture.
At this point can move on to immunofluorescence.
Protocol references
Acknowledgements
We thank the Lewis lab for helpful discussion.