Aug 29, 2024
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
External link: https://doi.org/10.1038/s41556-025-01712-y
Protocol Citation: Elias Adriaenssens 2024. Immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxynzwl8j/v1
Manuscript citation:
Adriaenssens, E., Schaar, S., Cook, A.S.I. et al. Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery. Nat Cell Biol 27, 1272–1287 (2025). https://doi.org/10.1038/s41556-025-01712-y
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 21, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 102632
Keywords: ASAPCRN, process of immunoprecipitation, immunoprecipitation, protocol, protocol detail
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details the process of immunoprecipitation.
Materials
- Pierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
- NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX Pageruler Prestained Protein LadderThermo Fisher ScientificCatalog #26616
- nitrocellulose membranes (RPN132D, GE Healthcare)
- Mini Trans-Blot Cell (Bio-Rad)
- SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) SuperSignal™ West Femto Maximum Sensitivity SubstrateThermo FisherCatalog #34096
- ChemiDoc MP Imaging system (Bio-Rad).
Lysis buffer:
| KCl | 100 mM | |
| MgCl2 | 2.5 mM | |
| Tris-HCl | 20 mM | |
| pH | 7.4 | |
| NP-40 | 0.50% |
Steps
30m
Collect the HeLa cells by trypsinization and wash the cell pellet with PBS once before cells are lysed in lysis buffer (100 mM KCl, 2.5 mM MgCl2, 20 mM Tris-HCl pH 7.4, 0.5% NP-40).
| KCl | 100 mM | |
| MgCl2 | 2.5 mM | |
| Tris-HCl | 20 mM | |
| pH | 7.4 | |
| NP-40 | 0.50% |
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
Protein concentrations of the cleared protein lysates are then determined with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher) and equal amounts are incubated with beads.
Precoat the beads with GST (negative control), NIX-GST, or BNIP3-GST as described in the protocol for the microscopy-based bead assay.
Incubate the HeLa cell lysates Overnight with precoated beads.
20m
In the morning, wash the samples five times in lysis buffer before the beads are either submitted for analysis by mass spectrometry or for analysis by SDS-PAGE and western blotting by resuspending the beads in protein loading dye, supplemented with 100 millimolar (mM) DTT, and boiled for 00:05:00 at 95 °C .
Note that for mass spec samples, the NP-40 is omitted from the washing buffer.
5m
Load the samples on 4-12% SDS-PAGE gels (NP0322BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).
Transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for 01:00:00 at 4 °C using the Mini Trans-Blot Cell (Bio-Rad).
1h
After the transfer, membranes are blocked with 5% milk powder dissolved in PBS-Tween (0.1% Tween 20) for 01:00:00 at Room temperature .
1h
Incubate the membranes Overnight at 4 °C with primary antibodies dissolved in the blocking buffer, wash three times for 5 min, and incubate with species-matched secondary horseradish peroxidase (HRP)-coupled antibodies diluted 1:10,000 in blocking buffer for 01:00:00 at Room temperature .
2h
Wash for 00:05:00 (1/3).
5m
Wash for 00:05:00 (2/3).
5m
Wash for 00:05:00 (3/3).
5m
Wash the membranes three times with PBS-T and processed further for western blot detection.
Incubate the membranes with SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) and imaged with a ChemiDoc MP Imaging system (Bio-Rad).