Jun 27, 2023
Halo-LC3B processing assay to assess autophagy
- 1Laboratory of James H. Hurley, University of California, Berkeley, CA
Protocol Citation: Xuefeng Ren 2023. Halo-LC3B processing assay to assess autophagy. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qexzvo5/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 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83241
Keywords: Halo-LC3B processing assay, autophagy, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details Halo-LC3B processing assay to assess autophagy.
Attachments
738-1854.pdf
115KB
Guidelines
Reference: DOI: 10.7554/eLife.78923
Materials
Buffers and reagents:
Growth media: DMEM medium with GlutaMAX containing 10% FBS and 10% Pen-Strep.
1x PBS
Lysis buffer:
| A | |
| 25 mM HEPES pH 7.5 | |
| 200 mM NaCl | |
| 2 mM MgCl2 | |
| 10% glycerol | |
| 1 mM TCEP | |
| 0.2% n-dodecyl-β-D-maltoside | |
| pierce protease inhibitors | |
| Benzonase |
DMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566016
Gibco™ Fetal Bovine Serum value heat inactivated (formerly USDA-approved in North America or qualiFisher ScientificCatalog #A5256801
Penicillin-Streptomycin (10,000 U/mL)Gibco - Thermo FisherCatalog #15140122
EBSS, calcium, magnesium, phenol redThermo FisherCatalog #24010043
Janelia Fluor® HaloTag® LigandsPromegaCatalog #GA1110
Gibco™ Trypsin-EDTA (0.05%) phenol redFisher ScientificCatalog #25-300-120
Pierce Protease Inhibitor TabletsThermo FisherCatalog #A32963
n-Dodecyl-B-D-maltoside (DDM)Gold BiotechnologyCatalog # DDM5
Benzonase® Nuclease Purity > 90%Merck Millipore (EMD Millipore)Catalog #70746-4
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX
Halo-LC3B processing assay to assess autophagy
Halo-LC3B processing assay to assess autophagy
1h 50m
1h 50m
Generating HeLa cells expressing HaloTag-LC3B using pMRX-IP-HaloTag7-LC3 from Mizushima lab (Addgene #184899; DOI: 10.7554/eLife.78923).
Seed HeLa cells at 100-150K cells/well in 12-well plate one day before.
Next day, incubate cells with complete DMEM medium and 50 nanomolar (nM) JF646 HaloTag ligand (Promega) for 01:00:00 , and then wash twice with 1xPBS. The non-starved samples can be harvested immediately by trypsinization (step 5).
1h
To induce autophagy by starvation, treat cells with EBSS buffer (Gibco) for desired period.
After the treatment, harvest the cells by trypsinization.
Wash the wells with 1x PBS.
Incubate the wells with 0.5 mL trypsin at 37 °C for 00:05:00 .
5m
Add 0.5 mL complete medium into each well.
Transfer cells into pre-chilled Eppendorf tube, spin 2000 x g, 00:05:00 .
5m
Aspirate off the liquid.
Resuspend the cell pellets in 30 µL of lysis buffer and incubate On ice for 00:30:00 .
30m
Centrifuge the cell lysate at 21000 x g, 00:10:00 . Transfer the cleared lysate into another tube, and measure protein concentration nanodrop spectrophotometer (Thermo Fisher).
10m
For each sample, load 20 µg clarified lysates onto NuPAGE 4-12% Bis-Tris Gel (Thermo Fisher).
For in-gel fluorescence imaging, the gel was immediately visualized with ChemiDoc MP imaging system (Bio-Rad) after SDS PAGE. Band intensities are acquired by exciting samples at 546 nm (mCherry signal) and 647 nm (JF646 HaloTag ligand signal).