Mar 21, 2025
Co-immunoprecipitation ER-phagy receptors
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2025. Co-immunoprecipitation ER-phagy receptors. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrkd1lpk/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: January 21, 2025
Last Modified: March 21, 2025
Protocol Integer ID: 119233
Keywords: ASAPCRN
Funders Acknowledgements:
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Abstract
This protocol details the co-immunoprecipitation of ER-phagy receptors.
Materials
pHAGE-TEX264-GFPaddgeneCatalog #201925 or pHAGE-FAM134C-GFPaddgeneCatalog #201927
Earle′s Balanced SaltsMerck MilliporeSigma (Sigma-Aldrich)Catalog #Earle′s Balanced Salts
Pierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
ChromoTek GFP-Trap® AgaroseProteintechCatalog # gta-20
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX
Lysis buffer
| A | B | |
| KCl | 150 mM | |
| MgCl2 | 2.5 mM | |
| Tris-HCl pH 7.4 | 20 mM | |
| NP-40 | 0.5% |
Washing buffer
| A | B | |
| KCl | 150 mM | |
| MgCl2 | 2.5 mM | |
| Tris-HCl pH 7.4 | 20 mM |
Procedure
Procedure
6h 35m
6h 35m
Wild-type HeLa cells are transiently transfected with pHAGE_TEX264-GFP (RRID:Addgene_201925) or pHAGE_FAM134C-GFP (RRID:Addgene_201927) using Lipofectamine 3000 (Thermo Fisher).
After seeding, treat the cells for 06:00:00 with Earle’s balanced salt medium starvation medium to induce ER-phagy.
6h
After the treatment, collect the cells by trypsinization and wash the cell pellet with PBS once before cells are lysed in lysis buffer.
Lysis buffer:
| A | B | |
| KCl | 150 mM | |
| MgCl2 | 2.5 mM | |
| Tris-HCl pH 7.4 | 20 mM | |
| NP-40 | 0.5% |
Lyse the samples for 00:20:00 On ice before cell lysates are cleared by centrifugation at 20000 x g, 4°C, 00:10:00 .
30m
Then, determine the protein concentrations of the cleared protein lysates with the Pierce Detergent Compatible Bradford Assay Kit.
Incubate the 6 mg of cell lysate Overnight with 20 µL of GFP-Trap agarose beads.
In the morning, wash the samples three times in washing buffer before the beads are resuspended in protein loading dye, supplement with 100 millimolar (mM) DTT, and boil for 00:05:00 at 95 °C .
Washing buffer:
| A | B | |
| KCl | 150 mM | |
| MgCl2 | 2.5 mM | |
| Tris-HCl pH 7.4 | 20 mM |
5m
Load the samples on 4-12% SDS-PAGE gels and analyze by western blotting as described above.