Jun 30, 2025
Immunoprecipitation from primary neurons
- Bishal Basak1,2,
- Erika Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
- University of Pennsylvania
External link: https://doi.org/10.1038/s41467-025-62379-5
Protocol Citation: Bishal Basak, Erika Holzbaur 2025. Immunoprecipitation from primary neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21m7pg1y/v1
Manuscript citation:
Basak, B., Holzbaur, E.L.F. Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy. Nat Commun 16, 7367 (2025). https://doi.org/10.1038/s41467-025-62379-5
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 30, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 221319
Keywords: immunoprecipitation from primary neurons protocol, immunoprecipitating protein, proteins from neuronal lysate, neuronal lysate, immunoprecipitation, primary neuron, primary neurons protocol, protein
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Protocol for immunoprecipitating proteins from neuronal lysates
Troubleshooting
Plate neurons
Plate 3- 5 million neurons in a 10 cm dish at days in vitro (DIV) 0
Immunoprecipitation steps
When neurons are mature (DIV 6 or more), treat neurons depending on experimental needs
Post treatment, aspirate out media, wash neurons once with 1X fresh PBS
Lyse neurons in lysis buffer containing 50 mM HEPES (pH=7.4), 1 mM EDTA, 1 mM MgCl2, 25 mM NaCl, 0.5% Triton X-100, 20μg/mL Leupeptin, 2 mM DTT, 20 μg/mL TAME, 2 μg/mL Pepstatin A, 1 mM PMSF, along with 200 nM of the deubiquitinase inhibitor N-ethylmaleimide.
Post lysis of the neurons, spin the lysate at 17000x g for 10 mins, and discard the pellet.
Store 5% of the supernatant to be used for western blotting later.
Divide the remaining supernatant into two equal parts- to one part add Antibody and to other part add equal amounts of the corresponding IgG control.
Incubate supernatants overnight with the antibody or its corresponding IgG at 40C in an orbital rotor.
The following day, wash 50 μl of Dynabeads Protein A (for Rabbit antibodies/IgG) (Invitrogen 10002D) or Dynabeads Protein G (for Mouse antibodies/IgG) (Invitrogen 10004D) per sample thrice with 1X PBS containing 0.02% Tween-20 followed by one final wash in the lysis buffer.
After washing, incubate the beads with the antibody or IgG containing supernatants for 4 hrs at 40C in an orbital rotor.
Post incubation, magnetically isolate the beads containing the IP-ed complex and wash 4 times with 1X PBS.
Elute IPed proteins in 2x SDS-containing sample buffer at 950C for 10 minutes, and use samples for western blotting.