Oct 27, 2023
Assessing autophagy using the HaloTag-LC3B cleavage assay
- 1Walter and Eliza Hall Institute of Medical Research
Protocol Citation: marvin.skul 2023. Assessing autophagy using the HaloTag-LC3B cleavage assay. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdo9zlmk/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: October 26, 2023
Last Modified: October 27, 2023
Protocol Integer ID: 89914
Keywords: lc3b during starvation autophagy, assessing autophagy, starvation autophagy, lc3b cleavage assay assay, lc3b, assay
Abstract
Assay to detect HaloTag-LC3B during starvation autophagy based on https://doi.org/10.7554/eLife.78923.
Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
9h 52m
Generate HeLa cells expressing HaloTag-LC3B using pMRX-IP-HaloTag7-LC3 from Mizushima lab (Addgene #184899; DOI: 10.7554/eLife.78923).
Seed 400-450K cells in a six-well plate one day prior to the experiment.
Feed cells with 1 mL of complete DMEM for 01:00:00
1h
Replace with 1 mL of complete DMEM containing 50 nanomolar (nM) TMR-conjugated Halo ligand (Promega, GA1120) and incubate at 37 °C for 00:20:00
20m
Wash cells thrice with 1x PBS followed by incubation in 2 mL of EBSS (Gibco, 24010043) for 06:00:00
6h
Following starvation, harvest cells via cell scraping On ice .
Aspirate off DMEM and wash cells in 1 mL of ice cold 1x PBS.
Scrape cells from each well in 600 µL of ice-cold 1x PBS and transfer into chilled 1.5 ml Eppendorf tube.
To collect residual cells, add another 600 µL of ice-cold 1x PBS transfer into 1.5ml eppendorf tube.
Spin down cells at 10000 x g, 4°C for 00:01:00 and aspirate off PBS.
1m
Perform a quick-spin at 10000 x g, 4°C and aspirate off residual PBS.
Cell pellets can be frozen at -20 °C or immediately used for immunoblotting
Lyse cells in 1 xLDS NuPAGE‱ LDS Sample Buffer (Invitrogen, NP0007) containing 100 millimolar (mM) DTT and Boil at 99 °C on thermomixer with shaking.
Measure protein concentration using A280 setting on Nanodrop. Ensure sample concentration is between 4-6 mg/ml.
Load 20 ug of protein on 4-12% Bis-Tris gels (Invitrogen, WG1402A). Run gel using 3-step ramp up setting (100v for 10mins, 150v for 10mins and 180v for 55mins).
Transfer protein using wet transfer method onto an Immobilon-P PVDF Membrane ( 0.45 µm pore size) using a Criterion blotter (Bio-rad, 1704070), 100v for 01:00:00 .
1h
Wash and block PVDF membrane:
- 1x in PVDF destain for 00:01:00
- 3x in TBS-T (1xTBS/0.05% Tween-20) for 00:02:00 each.
- Block in 10% Milk/TBS-T for 00:20:00
- 3x in TBS-T for 00:02:00 each
25m
Cut the blots at the 65kDa molecular weight marker and incubate in primary antibodies made up in TBS-T/3% BSA overnight at 4 °C on rocking platform
- >65kDa section in VCP (Cell signalling, 2648S) (1/1000 dilution)
- <65kDa section in anti-HaloTag (Promega, G9211) (1/100 dilution)
Following overnight incubation, recycle antibodies back into tubes and wash PVDF membrane.
- 3x in TBS-T for 00:02:00 each
2m
Incubate in anti-mouse HRP (Cell signalling, 7076S) and anti-rabbit HRP secondary antibodies (Cell Signalling, 7074S) for 01:00:00
- HaloTag (1/10,000 dilution for secondary)
- VCP (1/5,000 dilution for secondary)
1h
Wash blots prior to developing in ECL prime (Cytiva, RPN2232SK)
- 2x in TBS-T for 00:02:00 each.
- 2x in TBS for 00:02:00 each.
4m